The role of CYP 1A1 in the in vitro metabolism of pregnenolone by the liver of rainbow trout embryos.

The in vitro metabolism of pregnenolone (P5) was investigated using whole liver preparations taken from rainbow trout (Oncorhynchus mykiss) embryos sampled between 55 and 61 days post-fertilization. The intent of the study was to use HPLC techniques to separate and identify the metabolites of hepatic P5 metabolism and identify the enzyme(s) involved. The major metabolite of [3H]P5 catabolism was [3H]7alpha-hydroxypregnenolone ([3H]7alphaOHP5), and the enzyme involved was hypothesized to be a cytochrome P450 (CYP) isozyme. To test that hypothesis, whole liver preparations from embryos were pre-treated with selected CYP inhibitors prior to incubation with [3H]P5 and post-mitochondrial supernatant (PMS) fractions of embryo livers were pre-treated with specific antibodies raised against rainbow trout CYP 1A1 prior to incubation with radiolabelled steroid precursor. Three of the four inhibitors used (Miconazole, Clotimazole, Ketokonazole) and the CYP 1A1 antibodies totally blocked the conversion of [3H]P(5) to [3H]7alphaOHP5, and the fourth, Metyrapone, partially blocked the conversion. These results suggest that CYP 1A1 is the major enzyme involved in hepatic catabolism of P5 by rainbow trout embryos.

In vitro metabolism of pregnenolone to 7alpha-hydroxypregnenolone by rainbow trout embryos.

Tissues taken from rainbow trout embryos at several developmental stages, were incubated in the presence of radioactively-labelled pregnenolone in order to determine the capability of salmonid embryos to metabolize steroids, such as pregnenolone, that are incorporated into the oocyte during gonadal growth and maturation. High performance liquid chromatography was used to separate the steroid products, and gas chromatography-mass spectrometry was applied for the chemical identification of the product. 7alpha-Hydroxypregnenolone, previously known to be produced only by ovarian tissues, was found to be the sole metabolite of pregnenolone metabolism by rainbow trout embryos. Sulfate and glucuronide conjugated forms of 7alpha-hydroxypregnenolone were also produced. We hypothesize that this metabolite provides a pathway for excretion of pregnenolone, enabling the embryo to maintain its own steroid milieu, although the possibility of 7alpha-hydroxypregnenolone also playing a physiological role cannot be excluded.

Blood steroid profile and in vitro steroidogenesis by ovarian follicles and testis fragments of adult sea lamprey, Petromyzon marinus.

The main purpose of the study was to identify the principal gonadal steroids synthesized by male and female sea lampreys, Petromyzon marinus. To achieve this, we used high performance liquid chromatography to separate the steroids in the serum of sexually mature animals, and to separate the steroids produced by gonadal tissue incubated in the presence of radiolabelled precursor steroids, as a means of identifying the major steroidogenic pathways. We were unable to detect evidence of the 'classical' steroids, such as 17beta-estradiol (E(2)) or testosterone (T) in the serum of either male or female lampreys. Instead, the principal chromatographic peaks contained very polar compounds that had elution times consistent with 15alpha-hydroxylated estrogens and androgens, and there were sex-specific differences in the chemical nature and the quantity of these compounds. Testis fragments or ovarian follicles co-incubated with tritium-labelled pregnenolone ([3H]P(5)), 17-hydroxyprogesterone ([3H]17OHP(4)), or androstenedione ([3H]A(4)), provided additional confirmation that the gonads synthesize a range of very polar steroids, and the metabolites found were consistent with the presence of a 15alpha-hydroxylated (15alphaOH) metabolic pathway common to testis and ovary. For ovarian tissue, the major 'end product' metabolites from all three precursors were 15alphaOH-estrogens, and for testis tissue 15alpha-hydroxyprogesterone (15alphaOHP(4)) and 15alpha-hydroxytestosterone (15alphaOHT) and small amounts of 15alphaOH estrogen. Small amounts of E(2) were also produced by both ovarian (all substrates) and testicular tissue (some substrates). Although it was assumed that the E(2) was synthesized via the aromatization of T, [3H]T was not found as an intermediate metabolite. The study suggests that the principal gonadal steroids in sea lamprey are 15alpha-OH compounds, and that only small amounts of E(2) or T are synthesized by the gonads at this stage of reproductive development. There was no direct evidence of progesterone (P(4)) synthesis from [3H]P(5), although the metabolites synthesized by both testis and ovary were indicative of a metabolic pathway that involved P(4) as an intermediate.