PMC

Changes in the Zn concentrations in the testis of the Japanese eel after injection of human CG (hCG). The different letters indicate statistically significant differences (P < 0.05).

Light micrographs of testicular fragments after culture with 10 ng/mL KT and 0.001 mM TPEN for 15 days. (A) Control; (B) cultured with 10 ng/mL KT; (C) cultured with KT and 0.001 mM TPEN; (D) cultured with KT, TPEN, and 0.0025 mM ZnCl₂. SgA, type A- spermatogonia; SgB, type B-spermatogonia. (Scale bar: 20 μm.)

Effects of Zn chelators on the motility of Japanese eel sperm. (A) Ratio of motile sperm; (B) duration of sperm motility. C, control; Ca-1, incubated with 1 mM Ca-EDTA; T+Z, incubated with 1 mM TPEN and 1 mM ZnCl₂. Asterisks indicate statistically significant differences from the control.

Zinc distribution in germ cells of the Japanese eel. Zn was stained using ZnAF-2DA. Fluorescence images are shown for (A) Zn, and (B) mitochondria. (C) Bright field image of spermatogonia. (D) Zn fluorescence and (E) bright field image of spermatids and spermatozoa. M, mitochondria; St, spermatid; Sz, spermatozoa. (Scale bars: 10 μm.)

Light micrographs of testicular fragments after culture with Zn chelators for 6 days. (A–D) Hematoxylin and eosin-stained testicular fragments cultured for 6 days. (A) control; (B) cultured with 0.1 mM CaEDTA; (C) cultured with 0.01 mM TPEN; (D) cultured with 0.1 mM TPEN and 0.25 mM ZnCl₂. (E and F) One-day cultures of testicular fragments subjected to a TUNEL assay. (E) control; (F) cultured with TPEN. Dark stained cells are TUNEL-positive (E and F). Sg, spermatogonia; dSg, dead spermatogonia; T, TUNEL-positive cells. (Scale bar: 20 μm.)

The zinc distribution in the testis of the Japanese eel determined by staining with a Zn-specific fluorescent probe, ZnAF-2DA (A, C, and E). Bright field images are also shown (B, D, and F). (A and B) testicular fragments of the Japanese eel at 15 days after injection of hCG; (C and D) germ cells and Sertoli cells; (E and F) TPEN-treated germ cells. (Scale bars: A and B, 100 μm; C–F, 20 μm.)

Effects of a low dose TPEN upon germ cell proliferation in vitro. Testicular fragments were cultured with 0.001 mM TPEN and/or 10 ng/mL KT or DHP for 6 days (A) or cultured with TPEN and/or 1 ng/mL E2 (B). (C) Testicular fragments cultured without TPEN as a control for each steroid hormone; T, with TPEN; T+Z, with TPEN and Zn. KT, 10 ng/mL 11- ketotestosterone; DHP, 10 ng/mL 17α,20β-dihydroxy-4-pregnen-3-one; E2, 1 ng/mL estradiol-17β. Asterisks indicate significant differences from the negative control (P< 0.05). Daggers indicate significant differences from the control for each steroid hormone treatment (P < 0.05).

Effects of Zn and Zn chelators on the early stages of spermatogenesis in vitro. The BrdU-labeling index was determined for germ cells in testicular fragments cultured with Zn (A) or Zn chelators (B) with or without KT. The number of BrdU-positive germ cells is expressed as a percentage of the total number of germ cells. C, control; Zn, ZnCl₂; KT, 11- ketotestosterone; TPEN, N,N,N′,N′-tetrakis(2-pyridylmethyl)ethylenediamine; CaEDTA, ethylenediamine -N,N,N′,N′- tetraacetic acid, calcium(II), disodium salt, dihydrate. Results are given as the mean ± SEM. The different letters on the columns indicate statistically significant differences (P < 0.05).