Inhibin activity in charcoal-extracted spent culture medium of Sertoli cells in Primary culture (CSCCM) was monitored by following the temporal inhibition of serum levels of luteinizing hormone (LH) and follicle-stimulating hormone (FSH) in normal adult male rats. Subcutaneous injection of CSCCM reduced LH and FSH levels within 15 min and maintained the inhibition up to 10 hr. No selective suppression of FSH was observed. The model was used to assess inhibin activity in CSCCM from Sertoli cell cultures incubated with Sa-dihydrotestosterone (DHT), testosterone (T), and estradiol -17p (El) (5 pg/ml) for 24 hr. CSCCM from T and EZ treated groups suppressed gonadotropins while the group exposed to DHT failed to elicit any inhibition. DHT seems to inhibit the release of inhibin by Sertoli Cells.

Testosterone-oestradiol-binding globulin (TeBG) has been isolated from serum or plasma of several species using procedures that yielded highly purified protein, but which required multiple and tedious chromatographic steps. In this report we describe a procedure for the isolation of TeBG which involves two chromatographic steps: androgen affinity chromatography followed by anion-exchange high performance liquid chromatography (anion-exchange HPLC). The purity of the final product was confirmed by silver staining following fractionation on sodium dodecyl sulphate-containing polyacrylamide gels. The size heterogeneity and specific binding activity of TeBGs purified from human, rabbit, or bull serum (or plasma) by this technique was indistinguishable from preparations obtained by conventional chromatography. The present technique shortened the entire purification procedure to about 5 working days and yielded milligram quantities of highly purified protein. Bases on our experience with serum or plasma from the human, rabbit, and bull, this approach should be suitable for isolation of TeBG from a wide range of species.

Investigations were undertaken to demonstrate bioassayable GnRH-like activity in the spent nutrient medium of Sertoli Cells in Primary Cell Cultures (SCCM) using an in vitro bioassay procedure. The bioassay involved the use of intact pituitaries from day-12 male rats. Amounts of LH and FSH released into the incubation medium in response to varying doses of synthetic GnRH were measured by radio-immunoassays. SCCM was extracted with methanol and diethylether. The extracted SCCM was evaporated under a stream of nitrogen at 50 degrees C. The dry residue was re-suspended in the assay medium and centrifuged. The supernatant was used as the source of GnRH-like activity. The treated SCCM gave a dose response parallel to that given by synthetic GnRH. Boiling and trypsinization of extracted SCCM destroyed the GnRH-like activity. Addition of synthetic GnRH to different doses of extracted SCCM gave additive response.

Sertoli cells isolated from testes of 16-18 days old male rats were maintained in culture. Incubation media from these culture were pooled on day 7 and tested for its inhibin-like activity either with (SCCM) or after charcoal treatment (CSCCM) in castrated adult male rats. The assay was based on the tacit assumption that SCCM or CSCCM would specifically lower circulating blood serum levels of FSH. Subcutaneous (sc)injections of CSCCM at a dose level of 1 mg protein per rat, per day, x 3 days caused a specific suppression of FSH levels, while lower dosages of CSCCM (Protein content of 300 micrograms or 600 micrograms/rat/day, x 3 days) were without any affect on basal levels of FSH and LH. SCCM was ineffective at all dose levels tested. Intracardiac injections of varying doses of LHRH (25 to 400 ng/rat) to CSCCM pre-treated rats (200 micrograms/rat/day, x 3 days) failed to increase the levels of LH and FSH. These results support the presence of inhibin like activity in SCCM by a bioassay procedure alternate to in vitro pituitary cell culture system used by other investigators.

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