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As much as 70 % of the urinary metabolites of labelled progesterone in the rabbit consist of compounds which can be extracted from ether into aqueous sodium bicarbonate (Cooke, Rogers & Thomas, 1963). In view of the quantitative significance of this group of metabolites it was of interest to establish whether androstenedione also gave rise to bicarbonate-extractable metabolites.
Four mature Dutch female rabbits were injected subcutaneously with 0·15, 15, 25 and 50 mg. of [4-14C]androstenedione (8 μc) respectively, and urine was collected for 65 hr. The following procedures (designated as A, B and C in Fig. 1) were used for the hydrolysis of the steroid conjugates:
Procedure A.
In two experiments (when 0·15 and 15 mg. of steroid were injected) 15% (v/v) concentrated hydrochloric acid was added to the urine, and the solution was heated on a boiling water bath for 30 min.
Procedures B and C.
In the
Search for other papers by L. J. DONALDSON in
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Search for other papers by G. H. THOMAS in
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SUMMARY
DNA synthetic activity was monitored in rat and human prostate using [125I]iododeoxyuridine ([125I]UdR). Fresh prostate tissue from 6-week-old rats showed higher incorporation of [125I]UdR than that from 12- or 26-week-old rats. During culture for up to 6 days in the absence of hormones, the incorporation of [125I]UdR fell to a low level for all three age groups. Stimulatory effects were seen when rat prostates were cultured in the presence of insulin (3 μg/ml) and testosterone (10−7 mol/l), the incorporation on day 4 of culture being commensurate with that of fresh prostate of the corresponding age. Thus the magnitude of the response was higher for the 6-week-old prostate than that for the other two age groups. A similar age-related pattern of androgen stimulation was observed in experiments in which immature and adult castrated rats were injected daily with testosterone and the freshly removed prostates were incubated with [125I]UdR.
Although insulin, by itself, had a stimulatory effect on [125I]UdR incorporation in cultured prostate, the magnitude of the response did not differ in the 6- and 26-week-old prostate tissue. Maximal stimulation was obtained using 25 μg insulin/ml.
Tissue from a benign prostatic hyperplasia was also responsive to insulin in culture but it differed from rat prostate in that increased proliferative activity occurred even in the absence of hormone stimulation. This spontaneous surge in activity during culture tended to mask the stimulatory effects of insulin and testosterone at concentrations of 3 μg/ml and 10−7 mol/l respectively.
Search for other papers by P. M. DENNIS in
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Search for other papers by G. H. THOMAS in
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Δ5-3β-Hydroxysteroid dehydrogenase is a key enzyme involved in steroidogenesis, and has been demonstrated histochemically in the ovaries of a number of species (Baillie, Ferguson & Hart, 1966). The histochemical technique requires the addition of NAD and steroid substrate to the incubation medium, and the presence in the cellular structure of NADH2-dehydrogenase for the effective transfer of hydrogen to the tetrazolium dye (Wattenberg, 1958). Since monkey ovaries were available to us, it was decided to examine them for both NADH2-dehydrogenase and Δ5-3β-hydroxysteroid dehydrogenase. No previous reports have appeared on the localization of these enzymes in the ovaries of the monkey.
Six rhesus monkeys (Macaca mulatta) were investigated, two of which were pregnant (52 and 58 days p.c.). Only one of the pair of ovaries from each of the pregnant animals was available for this investigation. The non-pregnant animals were bilaterally ovariectomized on day
Search for other papers by J. C. MACARTNEY in
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SUMMARY
NADP-linked 17β- and 20α-reductase has been investigated in the uterus of immature (8–15 weeks) and adult rabbits using three labelled steroid substrates: progesterone, androstenedione and oestrone. The enzymic profile of the adult uterus differed from that of the immature, in that only the adult showed high levels of a 17β-reductase which had far greater specificity for oestrone than for androstenedione. Compared with the adult rabbit, the uterus of the rat, ferret and rhesus monkey showed little or no reductase activity.
Studies on the enzyme levels of the ovariectomized adult rabbit, and the immature rabbit treated with gonadotrophin, oestradiol, and oestradiol together with progesterone, showed that the uterine oestrone 17β-reductase was stimulated by the ovarian hormones; the response of androstenedione 17β-reductase to these hormone treatments was less marked. The oestradioldependant oestrone 17β-reductase was found predominantly in the myometrium.
Search for other papers by R. J. HEITZMAN in
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SUMMARY
The ketonic and non-ketonic neutral steroids from the urine of pregnant dairy cows were examined by gas chromatography. The free and acetylated metabolites were chromatographed on QF-1, the trimethylsilyl ethers on NPGA, and the oxidized material on QF-1, SE-30 and NPGA. As an aid to structural identifications, the elution ranges for various groups of metabolites were calculated for steroid alcohols and acetates on QF-1, and for trimethylsilyl ethers on NPGA. Epiandrosterone, aetiocholanolone and androsterone were the most abundant metabolites in the ketonic fraction, their combined concentration in the urine being of the order of 0·6 mg./l. Minor metabolites in the ketonic fraction included 5α- and 5β-androstanedione, 5β-pregnanedione and 3α-hydroxy-5β-pregnan-2-one. The non-ketonic fraction contained 5β-androstane-3α,17α-diol and 5α-androstane3β,17α-diol. In addition, evidence was obtained for the presence of a number of pregnanediols in this fraction, of which 5β-pregnane-3α,20α-diol was identified with certainty. The possibility of estimating this diol quantitatively by means of gas chromatography has been examined.
Search for other papers by SUSAN L. RUSSELL in
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SUMMARY
The metabolism and uptake of [2,4,6,7-3H]oestrone and [2,4,6,7-3H]oestradiol-17β were examined in explants of rabbit uterus, vagina and skeletal muscle in organ culture for 1 and 3 days. Irrespective of whether the uterus was cultured in the presence of [3H]oestrone or [3H]oestradiol-17β, the tissue contained predominantly [3H]oestradiol-17β. This was also true for vagina, but muscle showed only a limited ability to interconvert the two oestrogens.
The oestradiol-17β binding capacity of cultured uterus was also determined, and was shown to decline rapidly over the first 24 h of culture.
Search for other papers by J. M. CROCKER in
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SUMMARY
[6,7-3H]Oestrone was infused into virgin and 24-h post-partum lactating rabbits for a period of 1 h. At the end of the infusion period the concentration of free [6,7-3H]oestrone and [6,7-3H]oestradiol-17β was determined in plasma and in selected tissues (uterus, vagina, muscle, cerebral cortex, hypothalamus and anterior pituitary). Similar studies were carried out on virgin rabbits infused with [6,7-3H]oestradiol-17β. The tissues showed characteristic differences in the concentration of labelled oestradiol-17β and in the ratio labelled oestradiol-17β:oestrone, irrespective of whether the animal was infused with tritiated oestrone or oestradiol-17β. The uterus and vagina contained predominantly oestradiol-17β, while the oestradiol-17β:oestrone ratios of the pituitary and other tissues approximated to that of the plasma.
Search for other papers by I. R. SENCIALL in
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Search for other papers by G. H. THOMAS in
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SUMMARY
[4-14C]Progesterone, [4-14C]20α- and 20β-hydroxypregn-4-en-3-one were injected intravenously into deeply anaesthetized rabbits and the bile and urine collected for 6 h. Neutral metabolites recovered after Ketodase hydrolysis of bile accounted for a mean of 82·8% (range 68·0–98·8%), 68·4% (61·0–80·0%) and 59·0% (57·0–61·0%) of the metabolites of progesterone, 20α-and20β-hydroxypregn-4-en-3-one, respectively. Acidic metabolites accounted for no more than 14·7% of the biliary radioactivity. Similar recoveries of neutral metabolites were obtained after hydrolysis of bile with 15% HCl, but only 41·0% (22·2–62·0%), 45·3% (36·4–55·4%) and 13·7% (10·6–16·9%) of the urinary metabolites were extractable as neutral metabolites. Acidic metabolites represented a further 33·9% (28·9–49·5%), 34·9% (27·5–40·4%) and 38·6% (29·4–47·8%) of the urinary radioactivity, respectively.
Neutral ether soluble metabolites more polar than 5β-pregnane-3α,20α-diol were the most abundant metabolites. 3α-Hydroxy-5β-pregnan-20-one was identified in the bile as a metabolite of [4-14C]progesterone whereas 20α-hydroxypregn-4-en-3-one was present in the urine. 5β-Pregnane-3α, 20α-diol was identified as a biliary metabolite of [4-14C]20β-hydroxypregn-4-en-3-one.
Search for other papers by I. R. SENCIALL in
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Search for other papers by G. H. THOMAS in
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SUMMARY
Two rabbits were injected intravenously with [4-14C]progesterone and the bile and urine collected for 6 h. The crude bile was administered through an intraduodenal cannula to four rabbits as a single injection, and to three rabbits at a constant rate of infusion for 1·5 h. Bile and urine were collected hourly for up to 7 h. In the single injection experiments 42·3–64·4% of the radioactivity was excreted in the bile and 3·8–13·5% in the urine in 5–6 h. After constant rate infusion 35·4–64·8% and 4·7–9·5% of the radioactivity was excreted in the bile and urine respectively during 6–7 h. The hourly recovery of radioactivity in the neutral fractions of bile and urine decreased with time, whereas radioactivity not accounted for increased. Acidic metabolites represented an insignificant proportion of both biliary and urinary metabolites after enterohepatic recirculation.
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Search for other papers by ALMA M. COOKE in
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SUMMARY
After injection of [4-14C]progesterone into rabbits, 62% of the radioactivity recovered from the urine after hydrolysis is extractable from ether into sodium bicarbonate solution. Treatment of these acidic metabolites with potassium borohydride, followed by sodium bismuthate, yields neutral material which can be separated into three zones by thin-layer chromatography. The most polar zone, accounting for 61% of the radioactivity recovered from the plate, consists of epimers of 3,6-dihydroxyandrostan17-al.
