LIGHT AN ULTRASTRUCTURAL EVIDENCES OF HCG BINDING TO SERTOLI CELLS ISOLATED FROM Bufo arenarum

FORNES MW.; VINCENTI AE.; BURGOS MH.

Cancun

Simposio; ICEM14, Symposium II ? Electron Microscopy; 1998

ICEM

HCG injected into the dorsal lymphatic sacs produces the spermiation in Bufo arenarum (i.e., the appearance of spermatozoa in the cloaca) (1). The fine mechanics involved in this events is not clearly defined. But after the HCG injection, the Sertoli cells (SC) support an extensive vacuolization and the spermatozoa begin to be release (spermiation). To study the direct action and the binding of HCG on SC, we begun a series of in vitro experiments with isolated SC. The addition of HCG (200 IU, Endocorion®, Elea) to 2 ml of Sertoli cell suspension (SCS) (50 SC/ml) isolated from two testes produces an extensive vacuolization of SC with a complete losing of spermatozoa from its cavities (Fig.1). Instead, the control cells suspension (without HCG addition) maintain the sperm inside cavities without vacuolization (Fig.2). Some SCS were fixed (2% paraformaldehyde plus 0.1% glutaraldehyde in Holtfreter media(2)), dried on slide and assayed for the presence of HCG by immunocytochemical methodology using polyclonal anti HCG (1/40, Sigma) developed with a rabbit anti IgG (1/100 conjugated with peroxidase, Sigma Kit). The brown color on SC (dark SC on the plate in second page) marked the positive experiment (Fig.3) and the colorless cells marked the control cells (Fig.4). Finally, some SCS were also fixed and processed for TEM by conventional techniques. In this experiment the HCG was conjugates with colloidal gold (20 nm) following the Horisberger methodology (3). The SC in the presence of HCG showed and extensive vacuolization, lost of sperm with preservation of the microtubules (Fig.5) and the colloidal gold distributed on the surface and invesicles inside the SC (Fig.6). Instead, control SC treated with colloidal gold alone did not show any gold particle (data not show). We conclude that the HCG acts directly on Sertoli cells promoting the spermiation process and the HCG was incorporated in endocytic vesicles.