The ovine female reproductive tract experiences dramatic changes associated with season, stage of cycle, pregnancy and parturition. It is periodically exposed to foreign antigens such as those associated with sperm and the developing foetus and contributes to the complex, integrated hormonal network controlling reproductive function. The present investigations reveal that during changes in level of reproductive activity there are dramatic changes in the flow rate of lymph from the ovaries and uterus. There are relatively small changes in cell concentration so that cell output increases with flow rate. Large fluctuations in the levels of ovarian steroid hormones in the ovarian peripheral lymph reflect changes in steroidogenesis associated with folliculogenesis, ovulation, luteal function and luteolysis.
Cannulation of regional lymphatics and monitoring of the output and composition of lymph has facilitated study of the distribution of cellular components of lymph, the immune response of regional lymph nodes and the metabolic activity of various organs in changing physiological states (Hall and Morris 1962; Lindner et al 1966; Staples et al 1982). However, the relative ease with which lymphatics may be cannulated and the stoic nature of sheep, should not lead one to assume that such cannulation is necessarily physiological. The initial relative and absolute increase in neutrophils in the first two days after cannulation and the presence of MGC’s in lymph in the weeks following surgery suggest that acute and chronic inflammation is present. This inflammation results from the trauma of surgery and the presence of an indwelling cannula. In addition, the chronic drainage of lymph from a particular region would be expected to exert an effect on lymphocyte recirculation. The consequences of confinement on sheep also needs to be addressed as environmental stress has been shown to alter the immune responses of mice and pigs (Kelley et al 1984) and to affect the differential blood counts in several domestic species (Benjamin 1978). Sequential monitoring of corticosterone levels and indicators of cell and humoral immunity in sheep during their movement from free range to laboratory conditions may be a useful starting point in the evaluation of the effects of stress on the functioning of the ovine immune system. Systems designed for collection of lymph from free-ranging sheep, such as that described for the cow (Hein et al
1988), require further investigation. The recognition of areas of uncertainty associated with the chronic cannulation of lymphatics in no way negates the contributions which have been and which will continue to be made by the
Chapter Nine 144 utilization of this technique. Standardization of procedures and the reduction of artifactual changes will ensure that experiments based on lymphatic cannulation will continue to provide significant information on the functioning of the immune system and the contribution of the lymphatics to physiological processes.
There is little evidence in sheep that foetal antigens gain access to the maternal circulation. There is no measurable immune response to pregnancy in the regional lymph nodes and normal reactivity is maintained between foetal and maternal cells (Miyasaka and McCullagh 1981, 1982).* Furthermore, maternal lymphocytes which circulate through the pregnant uterus undergo no demonstrable diminution of their immune reactivity. Thus if foetal antigens do enter the maternal circulation they are masked in such a way as to make them undetectable by the maternal immune system and/or by conventional experimental technology. Lymphocyte subpopulations, as indicated by mAb labelling, in utero-ovarian peripheral lymph were determined (Section 6.3.a). The identification of cell types and particular cell subpopulations with mAb emphasized the selectivity involved in WBC migration through tissues. The failure to detect any significant alteration in lymphocyte subpopulations in utero- ovarian peripheral lymph between pregnant and nonpregnant ewes was interesting. This finding does not necessarily indicate that the ewe does not respond immunologically to the foetus, but does support the proposition that uterine lymphatics do not receive a contribution from the uterine lumen and endometrium. It is, however, also possible that an immunological response is occurring and that we are currently unable to recognize it. Unfortunately, the concept of "peripheral sensitization", as suggested by Brent and Medawar (1967), has received little attention. There is evidence of a change in the proportion of lymphocytes in the peripheral blood labelled by SBU-T4 and SBU-p220. The significance of these alterations is unknown.
The experiments which traced the movement of India ink injected into various layers of the uterus indicated that India ink was not transported from the uterine lumen to the draining lymphatics. However, this experiment did not address the possibility that the India ink may have left by way of blood vessels. This point may be investigated by a further study using a technique with greater sensitivity. One possibility would be the placement of radiolabelled material into the uterine lumen and subsequent autoradiography of tissue from such organs as the uterus, draining lymph nodes, spleen, lungs and Peyer’s patches. The search for trophoblast antigens in these tissues by the application of immunohistochemical techniques may also provide valuable information. However, there is no evidence of down regulation of the mother’s immune
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responsiveness as might be expected if foetal antigens gained access to the mother by the intravenous route (Greene, Sugimoto and Benacerraf 1978).
It is possible that the maternal immunological response to pregnancy in the sheep, occurs primarily at the maternal-foetal interface. What mechanisms might be operable at the maternal-foetal interface? Several mechanisms have been proposed and it is almost certain that the immunoregulation is multifaceted. The areas that currently appear worthy of further investigation in the sheep are: the location and structure of MHC I antigens on trophoblast; the association between trophoblast growth and maternal recognition; and the immunoactive products of the trophoblast and the decidua. The mechanisms involved in the immunoregulation of pregnancy in sheep are probably different to those in humans and rodents. The differences in gestation lengths and the type of placentation discourage the indiscreet transfer of concepts from one species to another. However, it is apparent that in many species the trophoblast produces pregnancy-specific proteins; to these has been assigned a role in the maternal recognition of pregnancy and prevention of luteolysis. The possibility that they may also have a role in regulation of the maternal immune response to pregnancy has not been exhaustively investigated.
High levels of progesterone (2000 ng/ml), similar to those found in the utero-ovarian peripheral lymphatics during the luteal phase of the oestrous cycle, were found to suppress the response of utero-ovarian peripheral lymph cells in
vitro to the mitogen Con A. However, as discussed in Chapter 1, progesterone is
unlikely to play a major immunosuppressive role in pregnancy as exogenous administration of progesterone is able to maintain pregnancy in ovariectomized ewes (Bindon 1971; Trounson and Moore 1974; Miller and Moore 1976).
A difference between the species was highlighted by the finding that the o I
enzyme Mg^ -ATPase is associated with T cells in sheep, in contrast to the situation in humans were it is found in B cells and not T cells (Muller-Hermelink 1974; Harigaya et al 1979; Palestro et al 1982; Crockard 1984). Histochemical techniques have a number of advantages over techniques using mAb. The materials required for the demonstration of histochemical markers are relatively inexpensive and widely available. Little manipulation of tissues and cells is necessary and the choice of fixative is generally not as crucial for histochemical techniques.
The importance of conducting cytological assessment of cells in conjunction with other forms of cell typing was reinforced by the present study. The monitoring of WBC differential counts for cells in utero-ovarian peripheral lymph provided data which could be used to monitor the sterility of the preparation, enabled a comparison with the percentage of lymphocytes found
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within the electronic window of the FACS, afforded a comparison with the percentage of macrophages identified histochemically and allowed revision of previous cytological findings. With respect to the last point, the recording of cell output, concentration and WBC differentials for utero-ovarian peripheral lymph in the current work, demonstrated a higher level of eosinophils than those reported by previous workers (Smith et al 1970b; Staples et al 1982; Hein 1985). Eosinophilic infiltration of the uterus and ovaries at various stages of the reproductive cycle (Gleich and Adolphson 1986; Murdoch 1987) is likely to be responsible for the elevation of eosinophil numbers in utero-ovarian peripheral lymph relative to peripheral lymph from other regions. In addition, this work contains the first report of the dramatic increase in RBC and WBC numbers in utero-ovarian peripheral lymph around the time of ovulation. This alteration in the cellular composition of the lymph probably results from the inflammatory process thought to be associated with ovulation (Espey 1980; Guraya 1985). The analysis of lymph plasma collected during this period may also allow the identification of inflammatory mediators or their breakdown products.
Ovarian follicles and young corpora lutea have a poor or absent lymphatic network (Morris and Sass 1966). Despite this, the lymphatics would still appear to reflect more accurately the milieu of tissue cells than efferent blood and the determination of the levels of various sex steroids in ovarian lymph collected around the time of ovulation provided some interesting results. Steroids, such as androstenedione, which were undetectable in the utero-ovarian peripheral lymph and peripheral blood of ewes, were found in significant quantities in ovarian lymph about the time ovulation was expected to take place. This is the first time that androstenedione and testosterone have been detected in ovarian lymph of sheep. The quantification of these steroids made possible an assessment of the activity of various enzymes associated with steroid metabolism in the ovary and this provided an insight into the rapid changes occurring in the ovary during the ovulation and subsequent luteinization. Clearly, observations on steroid levels in ovarian lymph give a more precise picture of changes in ovarian steroidogenesis than levels in peripheral blood plasma and should aid in determining the mechanisms by which the induction and inhibition of enzyme systems is accomplished.
Determining the composition of utero-ovarian peripheral lymph is not only important in itself, but also provides an insight into the activities of the uterus and ovaries. It has been stressed throughout this thesis that not all parts of the uterus and ovary have a lymphatic drainage. Nevertheless, the data obtained from the study of utero-ovarian peripheral lymph has furthered our knowledge of the workings of these organs.
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