Fig 1: Immunolocalisation of proteins identified as affected by sewage sludge exposure in the fetal sheep ovary. Heat-shock proteins, HSP60 (A), HSP70 (B), HSP90 (C) and HSPA4L (D) were all predominantly oocyte-specific, with strong cytoplasmic staining in most oocytes (blue arrows) but not all oocytes (white arrows). The fifth heat-shock protein, HSF1 (E) was localised in granulosa cells, pre-granulosa cells and many (but not all) somatic cells around the follicles. At higher power, HSF1 expression was detected in the nuclei of granulosa cells (blue arrows), but not in all pre-granulosa cells around primordial or forming primordial follicles (white arrow). CDKN1B (F) was localised in the cytoplasm and/or nuclei of some but not all oocytes and in the nuclei of some granulosa cells (blue arrows). Atretic follicles were CDKN1B-negative, but so were some healthy oocytes and follicles (white arrows). DNASE1 (G) showed intense staining in somatic cells around blood vessels and mesenchymal remnants (blue arrow) although all oocytes were negative (white arrow). ANXA1 (H) exhibited quite wide-spread staining, especially in the ovarian surface epithelium, the cytoplasm of many oocytes and scattered somatic cells (blue arrows). GSTM3 (I) was also principally localised to oocyte cytoplasm (blue arrow) although some oocytes, particularly those showing signs of atresia or dark condensed nuclei were negative (white arrow). IDH1 (J) was localised mainly in oocyte cytoplasm (blue arrow) but also in some somatic cells, including some surface epithelium cells. Positive staining is brown (DAB), counterstained by haematoxylin (blue). The bars denote scale for each image separately. Blue arrows highlight immuno-positive cells and white arrows immuno-negative cells. Two magnifications, taken from different ovaries, are shown for each antigen, separated by a white line. Each antigen is bounded by a black box to simplify interpretation. In all cases IgG-negative slides incubated with non-immune serum of the appropriate species were characterised by an absence of brown stain (one inset panel for each antigen).