Leukocytes present in the gonad. The testicular and ovarian areas of the gonad at different stages of the second RC immunostained with G7 (a-e), the ultrastructure of testicular acidophilic granulocytes (f), the percentage of gonad acidophilic granulocytes (g), the ultrastructure of testicular macrophage-like cells (h) and lymphocyte-like cells (i) and RT-PCR analysis of M-CSFR, TCR-β and IgM-H genes, as appropriate markers of macrophages and T and B lymphocytes, respectively (j). The acidophilic granulocytes appeared in the interstitial tissue of the testis at spermatogenesis (a), post-spawning (b) and testicular involution (c,d). Note that they also appeared between the germ cells at post-spawning (b) and around the necrotic areas at testicular involution (d). Scattered acidophilic granulocytes were also observed in the interstitial tissue of the ovarian area (e). Heterogeneous granules fused to each other were observed in the acidophilic granulocytes closed to the testicular necrotic areas (f). Testicular cell suspensions (n = 3–5 fish/month) were immunostained with the G7 and then analyzed by flow cytometry (g). The macrophage-like cells in the interstitial tissue in the vicinity of Leydig cells at spermatogenesis stage (h). Lymphocyte-like cells in the interstitial tissue (i). Total mRNA was obtained to mix the same amount of each mRNA from 4–5 fish/sample (j). Scale bar = 100 μm (a), 25 μm (b-e), 1 μm (f), 0.5 μm (h), 0.3 μm(i). Different letters denote statistically significant differences between the groups according to a Waller-Duncan test. (arrow), G7 positive cells; (white arrows), granules fused to each other; MLC, macrophage-like cell; LLC, lymphocyte-like cell; L, Leydig cell; Sg, Spermatogenesis; S, spawning; PS, post-spawning; TI, testicular involution; NFD, no further development; PTI, peripheral testicular area in the involution stage; ITI, internal testicular area in the involution stage; O, ovary; G, gametogenesis and C-, negative control.