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joa12002-sup-0001-FigS1.tifimage/tif14509KFig. S1. Immunoreactivity (IR) for representative neuroendocrine markers was observed in 30-μm-thick cross-sections of 12-day-old chick ovaries. The neuroendocrine cellular population identified (arrowheads) showed positive IR for both rate-limiting enzymes, tyrosine hydroxylase (TH) and tryptophan hydroxylase (TPH), which incorporate the amino acids tyrosine and tryptophan into the catecholamine and indolamine synthesis pathways, respectively (A and B). A robust TH staining was observed in nerve bundles and fibres (open arrowheads) that innervate the ovarian medulla that extend into cortical regions, reaching the perifollicular space (A). A similar but not as abundant or intense population of IR nerve fibres and bundles was identified with the heavy-chain neurofilament (NF-H) antibody (C). Positive IR for NF-H, was also observed in the neuroendocrine cells of the immature chick ovary (C). In all cases, these cells were small (less than 10 μm in diameter) and were observed scattered throughout the ovary, as single cells or small groups of them, thus a diffuse system by their distribution. Though located in the deep medulla, and in the interfollicular stroma of the cortex, the neuroendocrine cells were more easily found in the ovarian cortex-medulla transition region of the ages studied. A representative image as a control of the immunolocalization of neuroendocrine markers is shown, where the primary antibody was absent from the reactions (D). The dotted line marks the approximate boundary between the ovarian cortex (C), containing the follicles (F) and the medulla (M), that at this stage of development no longer has a dividing basement membrane, nor is there a clear separation between the two. Scale bar = 50 μm.

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