Conversion of Progesterone by Rat Medial Basal Hypothalamic Tissue to 5α-Pregnane-3,20-dione

Abstract

Tissue slices of proestrous rat medial basal hypothalami were incubated with tritiated progesterone. After subcellular fractionation, approximately 90% of the recovered radioactivity in the tissue was present in the nuclear fraction. About a third to a half of the recovered nuclear radioactivity was characterized as 5a-pregnane-3,20-dione by isotope dilution techniques. The other half of the recovered nuclear radioactivity was similarly characterized as unchanged tritiated progesterone. The remainder of the radioactivity associated with the nuclear fraction appeared to be isopolar on TLC with the 3aand 3/3-hydroxy-5a-pregnan-20ones. (Endocrinology 89: 940, 1971) T PROGESTERONE plays a role in regulating ovulation is well documented, albeit the exact mechanisms by which progesterone modulates the hypothalamic-pituitary axis are not known (1-6). In recent years, the uptake and metabolism by target tissues of estradiol and testosterone have been carefully studied (7-11). with the hope that data obtained would aid in the elucidation of their mechanism of action. However, the uptake and metabolism of these sex steroids and in particular progestrone by the hypothalamus have not been so extensively documented. Received December 28, 1970. This investigation was supported by USPHS Grants 2-TO1-HD-00104-06 and 5-PO1-HD03352-03 from NICHD, Ford Foundation Grant 630-0505A, and a General Research Support Grant to the University of Wisconsin from NIH, Division of Research Facilities and Resources. The metabolism of these steroids in the hypothalamus and brain may play an important role in the modulation of the feedback controls involved with ovulation via a key metabolic step. A knowledge of the metabolism of progesterone would be helpful in elucidating the nature of the reacting steroid species. In the present communication, we report on the in vitro metabolism of H-progesterone by the medial basal hypothalamus of proestrous female rats. The results indicate an appreciable portion of the radioactivity is associated with the nuclear fraction after subcellular fractionation, and the recovered nuclear radioactivity is largely in the form of progesterone and 5a-pregnane-3,20dione. Materials and Methods Materials. l,2-H-progesterone (SA 100 iiCi/ng) was purchased from New England Nuclear Corp. September 1971 NOTES AND COMMENTS 941 Radiochemical purity was ascertained by thinlayer chromatography (TLC) (ethyl acetate: CHC13; 1:13). Radioactivity was determined on a Packard Tri-Carb scintillation spectrometer (model 3003 or 3320). All other steroids were obtained from Sigma and found to be pure by thin-layer and gas-liquid chromatography (GLC). Silica Gel G (Brinkman Instruments, Inc.) was used for TLC. Incubation. Medial basal hypothalami (MBH) were removed from 10 proestrous rats (Holtzman Co.) between 7:00 and 9:00 AM CST (12). The tissue was washed with ice-cold 40 mM phosphate buffer (pH 7.2), cut Into 8 small pieces or slices, and incubated with 1.7 X10 dpm of H-progesterone and 1 ml of medium 199 (Microbiological Associates) in a Dubnoff metabolic shaker for 20 min at 37 C with an atmosphere of 95 % O2 and 5 % CO2. Following incubation the medium was removed, tissue washed with two 1 ml portions of medium 199, and homogenized in 1 ml of medium 199. The homogenate was centrifuged at 1000 Xg for 10 min to pellet the nuclear fraction (13). As a control, a similar preparation of 10 MBHs was boiled in buffer for 20 min and subjected to the same procedure. Extraction procedure. The nuclear pellet was extracted twice with 80% ethanol and once with ethyl acetate. Media and supernatant fractions were extracted by making these solutions 80% v/v in ethanol and centrifuging at 2500 Xg for 10 min. The resulting pellet was then extracted twice with 80 % ethanol and ethyl acetate. The nuclear extract was applied to TLC plates (ethyl acetate:CHCl3; 1:13) and sections corresponding to reference progesterone and 5a-pregnane-3,20-dione eluted twice with 3 ml absolute ethanol and once with 3 ml ethyl acetate. The remainder of the plate was scraped in 1 cm sections and assayed for radioactivity. For identification of the 2 radioactive zones isopolar with progesterone and 5a-pregnane-3,20-dione, isotopic dilution studies were carried out, as described in Table 2. In another run, the sample after 2 successive TLCs was put on GLC, the eluate trapped and counted to determine if the elution of mass and radioactivity were congruent. Gas-liquid chromatography. GLC was conducted on a Hewlett-Packard F and M Scientific (model 402) gas chromatograph equipped with a hydrogen flame detector and a 6'X3 mm Pyrex column packed with either 3% QF-1 or 3% SE-30 on 80/ 100 mesh gas chrom Q. GLC was done at 265 C.