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In introduction there is a brief review of the literature and an outline of the work embodied in the thesis. The thesis is divided into four main parts. Part 1. Gametogenesis and early development in Gigantocotyle bathycotyle (Fischoeder 1901), Nasmark 1937. This includes a description of the genitalia, with particular reference to the female organs and their associated ducts, an account of gametogenesis, egg shell formation and the early cleavage divisions. Part 2. The species of the genus, Paramphistomum , Fischoeder 1901, which occur in the British isles, with notes on some material from the Netherlands and France. Two new species of Paramphistomum are described and they are compared with Paramphistomum cervi (Zeder 1790 ) Fischoeder 1901 hitherto believed to be the only mammalian paramphistome occurring In this country Gametogenesis and early development are described briefly and compared with the processes in Gigantocotyle bathycotyle. The species obtained from the Netherlands and France are noted. Part 3. The development of the miracidium of Paramphistomum hiberniae from the time of deposition of the egg, until hatching. Observations are based almost, entirely on living material as only comparatively small numbers of eggs were available. The description of the miracidium includes notes on the staining reactions of various vital dyes and on the use of poly-vinyl alcohol as a means of keeping the miracidium still, without distortion. The process of hatching is described in some detail. Part 4. The attempts made to infect snails experimentally with paramphistome miracidia. An account is given of the collection and culturing of the eggs. Many hundreds of snails of various species were exposed to paramphistome miracidia. Results so far have been negative.

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10mM sodium fluoride inhibited the output of prostaglandin (PG)F<SUB>2α</SUB> from Day-7 guinea-pig endometrium and the outputs of PGF<SUB>2α</SUB> and PGE<SUB>2</SUB> from Day-15 guinea-pig endometrium during 24h of tissue culture. The incorporation of [<SUP>3</SUP>H]-leucine into cellular and secreted proteins by Day-15 guinea-pig endometrium in culture was inhibited by over 90% of sodium fluoride (10mM). Thus, as far as endometrial PG and protein synthesis are concerned, the effects of sodium fluoride are similar to those of other protein synthesis inhibitors and provide further evidence that the increase in endometrial PGF<SUB>2α</SUB> synthesis at the end of the oestrous cycle is dependent on the stimulation of fresh protein synthesis by oestradiol acting on a progesterone-primed uterus. Over a shorter period of culture (1h), sodium fluoride (10mM) stimulated the outputs of PGF<SUB>2α</SUB>, 6-keto-PGF<SUB>1α</SUB> and PGE<SUB>2</SUB> from Day-7 guinea-pig endometrium. However, the other G-protein modulators, cholera and pertussis toxin, had no effect on the output of PGs from Day-7 or Day-15 guinea-pig endometrium in culture for up to 24h. Consequently, a toxin-insensitive G-protein may be involved in mediating PG release from guinea-pig endometrium. Sodium fluoride also increased the outputs of PGF<SUB>2α</SUB>, 6-keto-PGF<SUB>1α</SUB> and PGE<SUB>2</SUB> from the Day-7 and Day-15 guinea-pig uterus superfused <i>in vitro</i>. The sodium fluoride-mediated increase in PG output from the superfused Day-7 uterus was unaffected by removal of extracellular calcium but was prevented by the intracellular calcium antagonist TMB-8. The calmodulin inhibitors, W-7 and trifluorperazine, and the phospholipase C inhibitor, neomycin, had no effect on the sodium fluoride-stimulated increases in PG output from the superfused Day-7 uterus. Therefore, sodium fluoride appears to stimulate uterine PG output by releasing calcium from intracellular stores, and this release is not due to activation of PLC nor is it mediated by calmodulin.

This is an investigation of the development of connections between the lateral geniculate nucleus (LGN) and the visual cortex in the mouse, using both <i>in vitro</i> and <i>in vivo</i> techniques. Visual cortical layer 4 is the target of ingrowing feniculate axons. Using immunohistological labelling techniques, I monitored the pre and postnatal migration of cells destined for layer 4 from the ventricular zone to the cortical plate <i>in vivo</i>. It was shown that, although the first of these cells enter the cortical plate as early as embryonic day 16 (E16), migration continues and layer 4 is not fully in place until postnatal day 7 (P7). The development of connections between the LGN and the visual cortex was investigated using <i>in vivo</i> immunohistological and tract-tracing methods with the carbocyanine dye, DiI. The LGN is formed between E12-14 and geniculocortical fibres are present in the visual cortex by E17. The main emphasis of this study was the mechanisms which control the outgrowth of axons from the LGN. Using an organotypic co-culture system, I searched for evidence of long range growth-promoting or trophic interactions between LGN explants and the occipital cortex. This technique has been widely used, with great success, by Yamamoto et al. (1989 and 1992), Bolz et al. (1990), Molnar and Blakemore (1991) and Toyama et al. (1991) to investigate the growth of axons into the visual cortex. Blocks of embryonic LGN were cultured in serum-free medium, either alone or with slices of embyonic cortex or early postnatal ocipital cortex, frontal cortex, cerebellum, medulla or liver (the ages of the explants were selected on the basis of the previous <i>in vivo</i> study).

Follicle development in the mouse ovary has been studied using two experimental approaches: firstly, a novel culture system was designed to investigate the metabolism of individual follicles for the first time, and secondly, chimaeric mice and molecular biology techniques were employed to study follicle morphogenesis and the developmental relationships between the different ovarian cell types. Physiological studies of follicle metabolism have been limited by the absence of a suitable culture system which is capable of supporting normal follicle growth and maturation. A novel culture system was developed to provide a physiological model for studies of metabolism by individual mouse ovarian follicles. This system supports the growth of individual primary mouse follicles to Graafian stages <i>in vitro</i> over a period of 5 days and is the first model demonstrating apparently normal ovulation of <i>in vitro</i>-grown follicles in response to LH. Preantral follicles of c.180 microns diameter were micro-dissected from the ovaries of 4 week old mice using fine needles and cultured individually in 20 μl of medium under mineral oil in V-shaped wells of a microtitre plate for a period of 6 days. Medium was supplemented with 1IU/ml hFSH and 5% serum from hypogonadal (<i>hpg/hpg</i>) mice. After every 24 hours of culture, follicles were washed and transferred to wells containing 20 μl of fresh medium: samples of 5-10 μl were then taken from the previous well for the analysis of metabolites. During growth and ovulation in culture, follicles and oocytes were morphologically indistinguishable from those during culture, indicating that premature luteinization does not occur in this system. Approximately 60% of dissected follicles reach preovulatory sizes during culture, which is considerably greater than the maturation rate <i>in vivo</i>, indicating that atresia is not a pre-programmed event. Experiments demonstrated that FSH-stimulation is required for full antral development and LH-induced ovulation, confirming that FSH confers LH-responsiveness to the follicle.

Divergent selection based on the LH response to a 5μg dose of GnRH, has created two lines of sheep which differ in their ability to release gonadotrophins in response to a GnRH challenge in both male and female lambs. Significant correlated between line differences have also been reported in female reproductive performance. The aim of this project was to investigate the regulation of gonadotrophin secretion in animals from the two lines, and to elucidate the primary site of the selected difference/s. Physiological studies of adult ewes and prepubertal ram lambs demonstrated that despite similar peripheral steroid concentrations, endogenous and exogenously stimulated gonadotrophin secretion differed significantly between the two lines. Mean LH and FSH concentrations in the prepubertal male lambs were significantly higher in the High line than the Low line, due to the secretion of LH pulses of significantly greater amplitude by the High line ram lambs. Similarly, higher amplitude LH pulses were observed in the High line ewes during the follicular phase of the oestrous cycle. The age related changes in basal LH secretion in the ram lambs and the observation of significant differences in LH pulse amplitude in the adult ewes during the follicular phase of the oestrous cycle, when progesterone negative feedback is reduced, indicate that the effects of the between line difference in the regulation of endogenous LH secretion are regulated by gonadal negative feedback. However studies in prepubertal ram lambs demonstrated that the primary site of the selected difference was at the level of the hypothalamo/pituitary gland complex. Studies of the regulation of LH secretion by the hypothalamo/pituitary gland complex demonstrated that the High line lambs appeared to secrete significantly less GnRH than the Low line and that the pituitary glands of the High line were 5 fold more sensitive to GnRH than the Low line. Pituitary sensitivity encompasses a large number of variables, including gonadotrophe and GnRH receptor number, the intracellular events which follow receptor activation and the amount of releasable LH stored in the pituitary gland, the individual or combined effects of which could result in differences in pituitary sensitivity. Pituitary gonadotrophe number/size was studied indirectly as a function of pituitary gland weight. The pituitary glands obtained from the High line tended to be heavier than those obtained from the Low line, however this difference was not statistically significant. The pituitary glands of the High line were also found to contain significantly more GnRH receptors/mg of protein than the Low line. The importance of this difference with regard to pituitary sensitivity was questioned, however, following the demonstration that the between line difference in the magnitude of the LH response was maintained <i>in vitro</i> following either GnRH stimulated LH release or the direct stimulation of both the Ca^2+-calmodulin and Protein Kinase C second messenger systems. Examination of the pituitary stores of LH in the two lines demonstrated that the 5μg dose of GnRH used in the selection programme stimulated a maximal release of LH in both lines, and that the High line stored significantly greater quantities of releasable LH compared with the Low line. The results also indicated that the two lines may differ in their ability to synthesise LH in response to GnRH stimulation.

Rat Mast Cell Proteinase II (RMCPII) from mucosal mast cell was titrated into rat serum and the resulting serpin:enzyme complex (SEC) was purified by affinity chromatography on anti-RMCPII Sepharose 4B and by MonoQ anion exchange. The purified complex was used to raise polyclonal antibodies which, after cross-absorption against RMCPII Sepharose 4B, were specific for serpin and were used to affinity purify two rat serpin molecules (RSI and RSII) which inhibit RMCPII in rat serum. The kinetic and thermodynamic constants characterising the interaction between RMCPII and RSI and RSII are: k<SUB>ass</SUB> 2.2.x10<SUP>5</SUP> M<SUP>-1</SUP>s<SUP>-1</SUP> and 1.65x10<SUP>5</SUP>M<SUP>-1</SUP>s<SUP>-1</SUP> respectively; K<SUB>i</SUB>, 3.6x10<SUP>-10</SUP>M and 1.0x10<SUP>-9</SUP>M; k<SUB>diss</SUB>, 7.9x10<SUP>-5</SUP>s<SUP>-1</SUP> and 1.65x10<SUP>-4</SUP>s<SUP>-1</SUP>. Amino-terminal sequence analysis indicated that RSI and II were distinct, differing at the N-terminal residues and were products of the rat SPI-1 locus. Rat Mast Cell Proteinase I (RMCPI) from connective tissue mast cells cleaved both RSI and RSII and was not inhibited. Further antibodies were generated against RSI and II, and partially successful attempts were made to raise a monoclonal antibody against rat serpin in complex with Mouse Mast Cell Proteinase Ie. Two polyclonal antibody preparations, raised in rabbit and sheep, were used to develop an ELISA that was specific for rat serpins, although no assay developed in this work would differentiate RSI from RSII. The change in serpin concentrations in serum and perfused tissues during helminth infection was monitored with the ELISA. Serpins concentrations in control plasma were determined to be 3mg/mL. Helminth infection caused a significant (p< 0.001) increase in this serpin concentration by day 7 of infection.

No description available.