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Gen Comp Endocrinol
2011 Sep 15;1733:454-60. doi: 10.1016/j.ygcen.2011.07.003.
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Cytokine components and mucosal immunity in the oviduct of Xenopus laevis (amphibia, pipidae).
Jantra S
,
Paulesu L
,
Lo Valvo M
,
Lillo F
,
Ietta F
,
Avanzati AM
,
Romagnoli R
,
Bechi N
,
Brizzi R
.
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Most studies on the mucosal immunity in female reproductive tissues have been performed in mammals. In all species, apart from their reproductive strategies, immunity in the genital mucosa is required to defend the host against luminal pathogens. In this study we investigated the role of the innate immunity of the oviductal mucosa of Xenopus laevis, an amphibian characterized by external fertilization. In particular we examined the expression and localization of Interleukin-1β (IL1B), Macrophage migration inhibitory factor (MIF) and Interleukin-1 receptor type 1 (IL1R1) in different oviductal portions including an upper glandular region, an intermediate and a lower aglandular region (the ovisac). Tissues were examined by immunohistochemistry and western blot using polyclonal antibodies against human molecules. IL1B, MIF and IL1R1 were all shown in the three oviductal regions examined, albeit with a general increase towards the external environment. A substantial difference among the cytokine components was also observed mainly in the epithelium of the glandular and intermediate regions. Specifically, all three molecules were expressed by the luminal ciliated cells while only IL1R1 was present in the unciliated cells at the bottom of the epithelial ingrowths. The expression of IL1R1 in these cells appeared as a continuous layer separating the epithelium from the underlying tissues. While supporting the role of the innate immune system for host's defense against pathogens, the peculiar distribution of the cytokine components in the oviduct of X. laevis suggests novel immunologic strategies useful to assure gland secretion essential for egg formation and fertilization.
Fig. 1.
Schematic representation of the oviduct of X. laevis and cross-sections of the regions examined. (A) The whole oviduct includes three main portions: a pars recta, which collects the ovulated eggs from the coelomic cavity, a pars convolute, which represents the main body of the oviduct, and the ovisac, where eggs accumulate before oviposition. The shaded areas are detailed in B and C. (B) The different oviductal regions examined: a glandular region (GR) from the lowest tract of the pars convoluta, an intermediate region (IR), characterized by strong gland reduction and the aglandular ovisac. (C) Cross-sections of the regions examined and observed under light microscope (hematoxylinâeosin staining). (C I and II): glandular region showing pluristratified, tubular glands, whose cells are filled with granular product. The oviductal epithelium shows ridges covered by luminal ciliated cells. Narrow ingrowths (asterisks) alternate to the ridges and their bottom is lined by unciliated cells (arrowheads in C II). Arrows point to gland lumina. (C III and IV): Intermediate region characterized by strong gland reduction and higher ciliated folds along the luminal surface. The bottom of the ingrowths (asterisk) consists of unciliated epithelial cells (arrowheads in C IV). (C V and VI): The ovisac lacks glands and its epithelium is arranged in folds of various size and shape, where ciliated and unciliated cells form a single layer. Arrows point to blood capillaries. ct = connective tissue, gl = glands, lu = oviductal lumen, mu = muscular sheet.
Fig. 2.
Immunoreactivity (shown in red) to anti-human IL1B (A, D and G), IL1R1 (B, E and H) and MIF (C, F and I) polyclonal antibodies in X. laevis oviductal regions. (A–C) Glandular region: The epithelium reveals expression for the three cytokine components IL1B (A), IL1R1 (B) and MIF (C) in the apical region of the luminal ciliated cells (thin arrows), whereas only IL1R1 (B) was present in the unciliated bottom cells (large arrow in B). The duct of most glands showed an evident immunoreactivity mainly for IL1R1 and MIF (double arrows in B and C). Secretory cells of the glands were always unstained. (D–F) Intermediate region: In the luminal ciliated cells immunostaining for IL1B, IL1R1and MIF was slightly more intense and widespread than in the glandular region (thin arrows in D–F). An intense and diffuse immunoreactivity for IL1R1 was evident in the cells at the bottom of the epithelium (large arrows in E). The glandular ducts showed immunoreactivity mainly for IL1R1 and MIF (double arrow in F). (G–I) Ovisac: Immunoreactivity was intense and widespread in almost all the epithelial cells (thin arrows in G–I). Immunoreactivity for all three proteins examined was also present in the endothelium (arrowheads in G–I) and in the connective and muscle tissues (asterisks in G–I). Controls for antibody specificity (J–L): Pre-adsorption of anti-human IL1B and anti-human MIF antibodies with the specific recombinant human proteins, resulted in a significant reduction of immunoreactivity (compare J with G, for IL1B and L with I, for MIF). Specificity of the anti-human IL1R1 antibody (K) was confirmed by pre-adsorbing the antibody on BeWo cell cultures (compare K with B). Inserts in J–L show negative controls performed by substituting the primary antibodies with TBS: no staining was observed.
Fig. 3.
Western blot profiles to anti-human IL1B (A) and MIF (B) in oviductal tissues and spleen of X. laevis. 100 μg/lane for IL1B and 50 μg/lane for MIF of total protein lysates were run in parallel with human recombinant (rh) IL1B (2 ng) or rhMIF (5 ng). Human placenta (30 μg) was used as positive control for MIF. (A) The anti-human IL1B recognized a band with a molecular weight of approximately 17 kDa in X. laevis tissues, corresponding to that of rhIL1B. (B) Anti-human MIF showed a band of about 12.5 kDa, corresponding to the predicted monomeric rhMIF protein. The two bands of approximately 25 and 36 kDa are consistent with the dimeric and trimeric form of MIF, respectively. The position of the molecular weight markers are indicated. The secondary antibody was a rabbit anti-goat conjugated with horseradish peroxidase (HRP) and the reaction was revealed by a chemiluminescent substrate.
