A novel collagen-glycosaminoglycan (C-GAG) substrate was developed to overcome the optical opacity of a HATF nitrocellulose substrate and to provide a more physiological permeable substrate for cocultured Sertoli and spermatogenic cells. Cocultures were prepared on optically transparent C-GAG discs attached to a polyester mesh to facilitate handling. Sertoli cells displayed a cuboidal-to-columnar shape; a large number of spermatogonia and primary spermatocytes connected by intercellular bridges were associated with basolateral and apical surfaces of Sertoli cells up to 12 days after plating. Rat Sertoli-spermatogenic cell cocultures have been used for testing the effect of toxicants on rat spermatogenesis in vitro. In our initial studies, we tested the effects of the toxicant gossypol on spermatogenic cells cocultured with Sertoli cells on nonpermeable (plastic) and permeable substrates (HATF nitrocellulose) under both standard culture conditions and during perifusion after achieving a continuous electrical-resistant cell monolayer. A selective mitochondrial structural damage was observed in spermatogenic cells (spermatogonia and spermatocytes) but not in the coexisting Sertoli cells. This damage was time- (15–60 min) and dose-dependent (0.1–10µM) and developed more rapidly under perifusion conditions. Similar mitochondrial damage was reported in the intact animal but required higher concentrations (mg) and longer administration time (months) for detection. Studies are in progress to evaluate the effect of additional toxic chemical agents on functional properties of Sertoli and spermatogenic cells in cocultures prepared on various classes of C-GAG substrates.
Similar content being viewed by others
Abbreviations
- C-GAG,:
-
collagen type I-glycosaminoglycans
- C-C6S,:
-
collagen type I-chondroitin-6-sulfate
- C-H,:
-
collagen type I-heparin
References
BACCETTI, B., BIGLIARDI, E., BURRINI, A.G., RENIERI, T., and SELMI, G. (1986). “The action of gossypol on rat germincal cells.” Gamete Res. 13:1–7.
BURGESS, W.H. and MACIAG, T. (1989). “The heparin-binding (fibroblast) growth factor family of proteins.” Annu. Rev. Biochem. 58:575–606.
DYM, M. and FAWCETT, D.W. (1971). “Further observations on the numbers of spermatogonia, spermatocytes, and spermatids connected by intracellular bridges in the mammalian testis.” Biol. Reprod. 4:195–215.
ERICKSON, R.P. (1972). “Haploid gene expression versus meitic drive: the relevance of intercellular bridges during spermatogenesis.” Nature 243:210–212.
KIERSZENBAUM, A.L. (1974). “RNA synthetic activities of Sertoli cells in the mouse testis.” Biol. Reprod. 11: 365–376.
KIERSZENBAUM, A.L. and TRES, L.L. (1987). “An automated perifusion system for the study of rat spermatogenesis in vitro.” Ann. N.Y. Acad. Sc. 513:146–157.
KLEINMAN, H.K., McGARVEY, M.L., HASSELL, J.R., STAR, V.L., CANNON, F.B., LAURIE, G.W., and MARTIN, G.R. (1986). “Basement membrane complexes with biological activity.” Biochemistry 25:312–318.
MIETHING, A. (1990). “Intercellular bridges between germ cells in the immature golden hamster testis: evidence for clonal and non-clonal mode of proliferation.” Cell Tissue Res. 262:559–567.
OKO, R. and HRUDKA, F. (1984). “Gossypol-induced early and delayed effects in the seminiferous epithelium of the adult rat.” Contracept. Deliv. Syst. 5:335–356.
PARVINEN, M. (1982). “Regulation of the seminiferous epithelium.” Endocrin. Rev. 3:404–417.
PRASAD, M.R.N. and DICZFALUSY, E. (1982). “Gossypol.” Int. J. Androl. 5:53–70.
QIAN, S.Z. and WANG, Z.G. (1984). “Gossypol: a potential antifertility agent in males.” Ann. Rev. Pharmacol. Toxicol. 24:329–360.
TRES, L.L. and KIERSZENBAUM, A.L. (1983). “Viability of rat spermatogenic cells in vitro is facilitated by their coculture with Sertoli cells in serum-free hormone-supplemented medium.” Proc. Natl. Acad. Sci. USA 80:3377–3381.
TRES, L.L., SMITH, E.P., VAN WYK, J.J., and KIERSZENBAUM, A.L. (1986). “Immunoreactive sites and accumulation of somatomedin-C in rat Sertoli-spermatogenic cell co-cultures.” Exp. Cell Res. 162:33–50.
TRES, L.L., SMITH, F.F., and KIERSZENBAUM, A.L. (1991). “Spermatogenesis in vitro: methodological advances and cellular functional parameters.” Serono Symposia Publications from Raven Press 71:115–125.
TRES, L.L., CAHN, F., and KIERSZENBAUM, A.L. (1992). “Rat Sertoli-spermatogenic cell cocultures established on permeable collagen-glycosaminoglycan crosslinked copolymers.” J. Tiss. Cult. Meth. in press.
SHABANOWITZ, R.B., DePHILIP, R.M., CROWELL, J.A., TRES, L.L., and KIERSZENBAUM, A.L. (1986). “Temporal appearance and cyclic behavior of Sertoli cell-specific secretory proteins during the development of the rat seminiferous tubule.” Biol. Reprod. 35:745–760.
UEDA, H., TRES, L.L., and KIERSZENBAUM, A.L. (1988). “Culture patterns and sorting of rat Sertoli cell secretory proteins.” J. Cell. Sci. 89:175–188.
YANNAS, I.V. (1990). “Biological active analogues of the extracellular matrix: artificial skin and nerves.” Angew. Chem. Int. Engl. 29:20–35.
ZHUANG, L.Z., PHILLIPS, D.M., GUNSALUS, G.L., BARDIN C.W., and MATHER, J.P. (1983). “Effects of gossypol on rat Sertoli and Leydig cells in primary culture and established cell lines.” J. Androl. 43:336–344.
Author information
Authors and Affiliations
Rights and permissions
About this article
Cite this article
Tres, L.L., Cahn, F. & Kierszenbaum, A.L. Permeable collagen-glycosaminoglycan cross-linked copolymers for the study of biological responses of coculturede Sertoli and spermatogenic cells. Cell Biol Toxicol 8, 61–71 (1992). https://doi.org/10.1007/BF00130512
Issue Date:
DOI: https://doi.org/10.1007/BF00130512