© 1993 Oxford University Press
research-article |
Identification of Stage-Specific Changes in Protein Secretion by Isolated Seminiferous Tubules from the Rat Following Exposure to Either m-Dinitrobenzene or Nitrobenzene
*MRC Reproductive Biology Unit, Centre for Reproductive Biology 37 Chalmers Street, Edinburgh EH3 9EW, Scotland
ICI plc, Central Toxicology Laboratory, Alderley Park Macclesfield, Cheshire SK1O 4TJ, England, United Kingdom
Received November 4, 1992; accepted May 13, 1993
The objective of this study was to identify effects of two known Sertoli cell toxicants on the secretion of proteins by seminiferous tubules (ST) isolated from adult rats at different stages of the spermatogenic cycle and cultured in vitro for 24 hr with [35S]methionine Adult rats received a single oral dose of 50 mg/kg metadinitrobenzene (m-DNB) or 300 mg/kg nitrobenzene (NB). Long lengths of ST at stages II–V, VI–VII or IX–XII were then isolated from control and treated rats at 1 or 3 days post-treatment; selection of stages was based on the stage specificity of the early (24–72 hr) adverse effects of m-DNB and NB on spermatogenesis in vivo. In addition, ST at the same stages were isolated from untreated rats and cultured in the presence or absence of m-DNB or NB (10–4 M). Incorporation of [35S]methionne into secreted proteins was assessed and the pattern of protein secretion evaluated using two-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis (2-D SDS-PAGE). ST isolated from rats pretreated 24 hr earlier with NB in vivo showed a significant decrease in the overall incorporation of [35S]methionne into secreted proteins at stages VI–VIII and IX–XII, whereas ST at stages II–V showed no such change; comparable protein changes were observed when 10–4M NB was added in vitro for 24 hr to ST isolated at the same stages from untreated rats. Similar results were obtained when ST protein secretion was evaluated 72 hr after treatment with NB in vitro or after the addition of NB in vitro to isolated ST from untreated rats for 24 or 72 hr. In general, the results obtained after exposure to m-DNB were comparable to those observed for NB, with the exception that the incorporation of [35S] into secreted proteins by ST at stages II–V tended to be increased by m-DNB exposure. Analysis of ST-secreted proteins by 2-D SDS-PAGE identified 6 "marker proteins" (MP) which showed major reproducible changes in secretion following exposure to either m-DNB or NB. In most cases (MP–1, –2, –3, –5, and –6) their secretion was reduced markedly; two of these proteins (MP–2 and MP–3) are secreted almost exclusively at stages VI–VIII and correspond in molecular size and pI to two recently reported androgen-regulated proteins which are thought to be products of the Sertoli cell. Exposure to either NB or m-DNB also resulted in the appearance of one protein (MP–4) which was not secreted in detectable amounts by ST from control animals. Previous data has shown that by 24 hr after administration of either m-DNB or NB to rats there is massive loss/degeneration of pachytene spermato cytes at stages VI–XII, whereas stages II–V of the spermatogenic cycle are not affected until 72 hr. The presently observed protein changes therefore either precede (stages II–V) or accompany (stages VI–XII) germ cell degeneration. This suggests that they may have potential use as markers of early toxicant-induced damage and/or that changes in these proteins may mediate some of the adverse testicular effects of m-DNB and NB.