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- Author: R. V. SHORT x
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SUMMARY
A pool of 500 ml. normal equine follicular fluid was examined for the presence of Δ5 3β-hydroxy steroids. Neither pregnenolone nor 17α-hydroxypregnenolone could be detected (< 1 μg./l.), but there appeared to be a small amount of dehydroepiandrosterone (DHA) present (8 μg./l.). These findings suggest that in the Graafian follicle of the mare the conversion of pregnenolone to progesterone normally proceeds fairly rapidly, and the pregnenolone → 17α-hydroxypregnenolone → DHA → androstenedione pathway is probably of minor importance.
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SUMMARY
A comparison has been made between the steroids present in equine follicular fluid and luteal tissue. Follicular fluid was found to contain progesterone, 17α-hydroxyprogesterone, androstenedione, epitestosterone, 19-norandrostenedione, oestrone, oestradiol-17β and 6α-hydroxyoestradiol-17β, in confirmation of previous studies. On the other hand, luteal tissue contained large amounts of progesterone and 20α-hydroxypregn-4-en-3-one, and a small quantity of 17α-hydroxyprogesterone. No C18 or C19 steroids could be detected.
These findings are therefore interpreted as evidence in favour of a 'two-cell type' theory of steroid formation in the ovary. One type, possibly the theca interna cell, has the ability to convert progesterone into oestrogens; the other type, possibly the luteinized granulosa cell, has a diminished 17-hydroxylating ability and is unable to cleave the C21 side chain to make C18 and C19 steroids. However, it does have an active 20-reductase enzyme system present.
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SUMMARY
Steroid determinations were carried out in 2·3 ml. cyst fluid obtained from the ovaries of a woman with the Stein—Leventhal syndrome. The following concentrations were found (μg. steroid/100 ml. fluid): pregnenolone, < 2·0; progesterone, < 9·0; 17α-hydroxyprogesterone, 24·0; androstenedione, 139·0; 19-hydroxyandrostenedione, < 4·0; oestrone, < 2·0; oestradiol-17β, < 2·0; 17α-hydroxypregnenolene, < 2·0; dehydroepiandrosterone, 10·0; testosterone, < 4·0.
These results therefore confirm and extend those obtained in a previous study (Short & London, 1961). They strongly suggest that in this syndrome there is a defective 19-hydroxylation of androstenedione, rather than either a defective 3β-ol dehydrogenase enzyme system or an increased ovarian production of testosterone as has been suggested by other workers.
The possibility that such a defect might be caused by an X chromosome abnormality in some of the theca interna cells of the ovary is discussed.
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SUMMARY
The following steroids were estimated in each of twelve samples of follicular fluid collected from oestrous, luteal-stage and pregnant mares: progesterone, 17α-hydroxyprogesterone, androstenedione, epitestosterone, 19-norandrostenedione, oestrone, oestradiol-17β and 6 'α'-hydroxyoestradiol-17β. In ten of the samples oestradiol-17β was the major steroid present. In one sample taken from a pregnant animal oestradiol-17β could not be detected at all, and in its place large amounts of progesterone and 20α-hydroxypregn-4-en-3-one were found.
The larger follicles tended to have the higher steroid concentrations. In this series, there was no significant difference in concentration between oestrous and luteal-phase animals with respect to any of the steroids measured (P > 0·05); the reasons for this are discussed. It is concluded that the gonadotrophic hormones do not act by influencing any rate-limiting reaction in the conversion of progesterone to oestrogens.
Since the ratios of the various steroids seem to differ greatly between follicular fluid and luteal tissue, it is postulated that cell type may be the chief discriminant of the secretory activity of the ovary.
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SUMMARY
Using paper chromatographic techniques, it has been possible to identify the following steroids in the follicular fluid of the mare (results expressed as μg/1.): oestradiol-17β, 460 μg; oestrone, 34 μg; cortisol, 12 μg; progesterone, 124 μg; 17α-hydroxyprogesterone, 66 μg; epitestosterone, 24 μg; androstenedione, 136 μg. In addition to these seven compounds, 136 μg of an unidentified oestrogen-like substance and 52 μg of an unidentified 17-ketosteroid were also present.
This is the first time that epitestosterone has been identified as a natural product, and the possible significance of this compound is discussed. 19-Hydroxyandrostenedione, the postulated intermediate in the formation of oestrone from androstenedione, was not detected in this study, and some doubt is therefore cast on the theory that it is an essential intermediate in the biosynthesis of the ovarian oestrogens.
The presence of relatively large amounts of an unidentified polar oestrogen in follicular fluid suggests that it may be of some physiological significance, although at the present time no information is available on the biological activity of this compound.
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SUMMARY
A method is described for the chemical determination of progesterone in the peripheral venous blood of women, horses, cattle, sheep and pigs. The recovery rate of progesterone added to human plasma is 73 ± 4% (s.e.); in the cow, the recovery rate is 63 ± 1·9% (s.e.). In pregnant women approximately 25 ml. of plasma is sufficient for an assay, but in domestic animals the blood concentration of progesterone is much lower and a 500 ml. sample of plasma is usually required.
The method has been used for the determination of blood progesterone levels in both pregnant and non-pregnant domestic animals. In women progesterone has been detected as early as the 10th week of pregnancy, and preliminary results in pregnant cattle are given in the following paper [Short, 1958].
In addition, the method allows the simultaneous determination of two other related compounds found in peripheral blood: 20α- and 20β-hydroxypregn-4-en-3one.
The method is suitable for routine laboratory determinations; results can be obtained within 24 hr of receipt of a blood sample, and it is possible to carry out a number of assays in 1 day.
If small amounts of progesterone are incubated with whole ox blood at 37° C, there is a slow transformation (half-life approximately 7 hr) to 20β-hydroxypregn-4-en-3one.
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SUMMARY
The content of progesterone was determined in the peripheral venous blood of cows from the 32nd day of pregnancy until the day before calving. The level ranged from 0·74 to 0·98 μg progesterone/100 ml. plasma throughout the 32nd-256th day period, but thereafter a marked decrease was observed, and on the day before calving the level was 0·1–0·4 μg/100 ml. plasma.
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SUMMARY
Two mares were operated on during oestrus; in the first animal a mixture of [16-3H]pregnenolone and [4-14C]androstenedione was injected into the largest Graafian follicle; in the second animal, [4-14C]pregnenolone and [7-3H]androstenedione were injected into the follicle. By cannulating the ovarian veins it was possible to measure the rate of transfer of radioactivity from the follicular fluid into the ovarian vein. This ranged from 3 to 9% of the injected dose per hour. At the end of each experiment, 45–52 min. after the injection of the labelled precursors, the follicular fluid was aspirated. Negligible amounts of radioactivity were recovered from the rest of the ovary and from the 'conjugated' fraction of the ovarian vein plasma and follicular fluid. Within the follicle 6·2 and 12·2% of the injected pregnenolone were converted to progesterone. Further metabolism to 17 α-hydroxyprogesterone, androstenedione and oestradiol-17β was very limited. Oestradiol-17β isolated from the ovarian vein had a higher specific activity than that from the follicular fluid while the 3H: 14C ratios were similar in both compartments. In addition, the 3H:14C ratios of androstenedione and oestradiol-17β in the ovarian vein were similar. Therefore it seems likely that oestradiol-17β in the follicular fluid is derived mainly from an extra-follicular source, probably the theca interna cells.
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SUMMARY
The fluorescence reaction for progesterone described by Touchstone & Murawec (1960) has been used to determine the concentration of progesterone in nineteen samples of peripheral blood from pregnant women, and in seventeen samples of peripheral blood from women during the course of the menstrual cycle.
There was good agreement between the ultraviolet and fluorescent estimates of progesterone in all the samples from pregnant women.
The concentrations found during the follicular phase of the menstrual cycle were in general lower than those found during the luteal phase. In one woman who was sampled repeatedly during the course of a menstrual cycle, there was a well defined rise in the level of progesterone in the blood after the expected date of ovulation.
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SUMMARY
[4-14C]Progesterone (10 μc, 154 μg.) was injected intravenously into a Suffolk ewe on the 115th day of gestation, and again a few hours before parturition. Within 5 min. of the injection, only about 1% of the radioactive progesterone was present in the circulating blood. During the ensuing 25 min., the fall in concentration was approximately exponential, the estimated half-lives being 7·3 ± 1·2 min. at day 115 and 8·1 ± 0·6 min. just before lambing. The difference between these half-lives is not significant.
Subject to a number of assumptions, these half-lives may be used as a measure of the rate at which progesterone is metabolized in the body. It is concluded that there are no pronounced changes in the rate of progesterone production or metabolism during the course of pregnancy in the ewe.
