The brain is a steroidogenic organ that expresses steroidogenic enzymes and produces neurosteroids. Although considerable information is now available regarding the steroidogenic capacity of the brain, little is known regarding the steroidogenic pathway and relative contributions of astrocytes, oligodendrocytes, and neurons to neurosteroidogenesis. In the present study, we investigated differential gene expression of the key steroidogenic enzymes using RT-PCR and quantitatively evaluated the production of neurosteroids by highly purified astrocytes, oligodendrocytes, and neurons from the cerebral cortex of neonatal rat brains using specific and sensitive RIAs. Astrocytes appear to be the most active steroidogenic cells in the brain. These cells express cytochrome P450 side-chain cleavage (P450scc), 17alpha-hydroxylase/C17-20-lyase (P450c17), 3beta-hydroxysteroid dehydrogenase (3betaHSD), 17beta-hydroxysteroid dehydrogenase (17betaHSD), and cytochrome P450 aromatase (P450arom) and produce pregnenolone (P5), progesterone (P4), dehydroepiandrosterone (DHEA), androstenedione (A4), testosterone (T), estradiol, and estrone. Oligodendrocytes express only P450scc and 3betaHSD and produce P5, P4, and A4. These cells do not express P450c17, 17betaHSD, or P450arom or produce DHEA, T, or estrogen. Neurons express P450scc, P450c17, 3betaHSD, and P450arom and produce P5, DHEA, A4, and estrogen, but do not express 17betaHSD or produce T. By comparing the ability of each cell type in the production of neurosteroids, astrocytes are the major producer of P4, DHEA, and androgens, whereas oligodendrocytes are predominantly the producer of P5 and neurons of estrogens. These findings serve to define the neurosteroidogenic pathway, with special emphasis on the dominant role of astrocytes and their interaction with oligodendrocytes and neurons in the genesis of DHEA and active sex steroids. Thus, we propose that neurosteroidogenesis is accomplished by a tripartite contribution of the three cell types in the brain.

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Blood testosterone levels were measured by RIA and gas chromatography-mass spectrometry in the pregnant mare. They were found to increase from the very beginning of pregnancy, reaching peak values 10 times higher than the basal values at the seventh month and then to return to basal values by the week after parturition. Testosterone binding by plasma proteins was investigated in nonpregnant and pregnant mares throughout gestation. Equilibrium dialysis and gel equilibration methods did not reveal any blood specific testosterone-binding activity at any gestational stage. Hence, blood testosterone increase cannot result as in the pregnant woman or guinea pig, from an increase in blood specific binding activity but more probably results from an increased steroid production. It is suggested that the maternal gonads would be responsible for the testosterone increase in early pregnancy, during the period of PMSG production, and the fetoplacental unit for the subsequent increase.

Nucleotide sequence analysis of the complimentary DNAs (cDNA) and N-terminal amino acid sequence analysis have shown that clusterin is equivalent to sulfated glycoprotein-2 (SGP-2), testosterone-repressed prostate protein-2 (TRPP-2), and androgen-repressed protein (ARP) in the rat, as well as serum/seminal plasma protein, SP-40,40, in the human. In view of its widespread presence in various species, a specific RIA was established to quantify the tissue distribution of this protein. Rat clusterin is present in almost all organ tissues examined, including testis, epididymis, serum, liver, prostate, seminal vesicles, and uterus. Displacement curves generated using cytosols prepared from these organs were parallel to those obtained using purified rat clusterin and crude Sertoli cell-enriched culture medium. Immunoreactive clusterin was also visualized in these organ extracts by immunoblots. Studies on the tissue distribution of immunoreactive clusterin using RIA revealed that the concentration of clusterin in the epididymis of adult rats was 6- and 10-fold higher than that in the serum and testis, respectively and is 50- to 100-fold higher in the liver, spleen, kidney, brain, ventral prostate, seminal vesicles, and uterus. A study of the distribution of clusterin in various compartments of the epididymis indicated its concentration in the caput epididymis was almost 3-fold higher than that in the corpus and cauda epididymis. After orchiectomy, the concentrations of clusterin in the ventral prostate and seminal vesicles increased as much as 100- and 10-fold and peaked at day 4 after surgery, respectively; daily injection of dihydrotestosterone (DHT) beginning at day 3 after orchiectomy reduced the concentrations of clusterin and restored them to a normal level. A different pattern was noted in the epididymis after orchiectomy; the concentration of clusterin in the caput epididymis decreased with time; however, daily injection of DHT beginning at day 3 increased the caput epididymal clusterin concentration and restored it to a normal level. The concentration of clusterin was not altered in the corpus or cauda epididymis after castration and/or DHT administration. Also, the serum and liver clusterin levels did not change with time after orchiectomy. These observations suggest that clusterin will be a valuable marker to monitor the diverse effects of androgen withdrawal in the male reproductive tract. We conclude that clusterin may be a multifunctional protein in view of its broad tissue distribution and association with numerous physiological and pathological conditions.