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Free and sulphate-conjugated 16-unsaturated C19 steroids in human testis tissue
Biochimica ( 1 Elsevier
et Biophysics Acta, Scientific Publishing
316 (1973) Company,
251-255
Amsterdam
- Printed
in The Netherlands
BBA 56302 FREE
AND
IN HUMAN
AIM0
SULPHATE-CONJUGATED TESTIS
16-UNSATURATED
C,,
STEROIDS
TISSUE
RUOKONEN
Steroid Research Laboratory, Department University of Helsinki, SF-001 70 Helsinki (Received
March
26th,
ofMedico1 Chemistry, I 7 (Finland)
1973)
SUMMARY
Free and monosulphated 5a-androst- I 6-en-3x-01, 5a-androst- I 6-en-3P-ol and 5, I 6-androstadien-3/?-ol were identified and quantified in human cadaver testis tissue. Free and monosulphated steroid fractions were isolated by Sephadex LH-20 chromatography. Further separation and purification of the r6-unsaturated C,, steroids was achieved using silicic acid and hydroxyalkoxypropyl Sephadex chromatography. The steroids were finally identified by gas-liquid chromatography and gas chromatography-mass spectrometry. In the free steroid fraction the relative abundance of the r6-unsaturated C,, steroids was 5cc-androst-r 6-en-3j%ol > 5, I 6-androstadien-3/Gol > 5a-androst- I 6-en -3~01 and in the monosulphate fraction 5, I 6-androstadien-3fl-ol > 5z-androst- I 6-en-3/Gol > 5x-androst- I 6-en-3c+ol.
INTRODUCTION
5a-Androst- I 6-en-3cc-ol and 5a-androst- I 6-en-3p-ol were first discovered in boar testis tissue’. Later, Brooksbank and Haslewood” isolated 5a-androst-r 6-en-3a01 from the glucuronide fraction of human urine. Its urinary excretion is almost three times as great in men as in women 3. Adrenocorticotropic hormone (ACTH)4 administration increases urinary 5u-androst- I 6-en-3r-ol excretion in both sexes and human chorionic gonadotrophin3 administration has the same effect in normal men and in patients with the testicular feminization syndrome 5. Thus, it seems likely that both the testes and the adrenals are capable of secreting 5a-androst-16-en-3cc-ol or its precursors. The purpose of this study was to investigate the possible role of human testis in the production of 16-unsaturated C,9 steroids. Trivial and systematic 3/J-hydroxy-5-pregnen-2o-one; corticotropic hormone.
names: testosterone, 178-hydroxy-4-androsten-3-one; stigmasterol, (2@)-24-ethyl-5,2z-cholestadien-3~-ol;
pregnenolone, ACTH, adreno-
A. RUOKONEN
252 TABLE DATA Subject
I ON SUBJECTS STUDIED ~____ __~ Age Time interval (years i after death (h)
Weight of combined testes
Cause of death
(gl B.T. L.R. M.L.
24 29 41
30 5 ‘4
34.5 48.0 22.2
Cerebral
hemorrhage
cord Cerebral hemorrhage Rupture
of spinal
EXPERIMENTAL Materials
Testes were obtained from cadavers (3 subjects). Prior to analysis the testicular capsule was removed and the tissue sample weighed and subsequently stored at - 20 “C. Data on the samples are given in Table I. R@rence compounds 5x-Androst-r6-en-3a-ol was obtained from professor W. Klyne (Steroid Reference Collection, London, England). 5, rb-Androstadien-3fi-ol and 4, r6-androstadien-3-one were kindly supplied by Dr D. B. Gower (London, England). 5cr-Androst16-en-3/?-ol and 5cc-androst-r6-en-3-one were purchased from Ikapharm (RamatGan, Israel). Methods Both testes were combined for steroid determination. The tissue sample was carefully homogenized in acetone-ethanol (I : I, v/v) and chloroform-methanol (I : I, v/v) and the combined filtrates were evaporated. The residue was chromatographed on a 20-g Sephadex LH-20 column, and the free and monosulphate-conjugated steroid fractions were collected as described by Laatikainen and Vihko6. The free steroid fraction was dried in vacua and partitioned between ethyl acetate and 0.01 M NaOH. The ethyl acetate phase was dried with MgSO,, evaporated and dissolved in 2- and I -ml portions of I 2 “/, ethyl acetate in toluene and applied to a 5-g silicic acid column (0.8 cm x 18. I cm) packed in toluene. The i6-unsaturated C,, steroids were eluted from the column with 40 ml of 12 % ethyl acetate in toluene. The final purification was carried out on a 2.5-g column (0.6 cm x 18.4 cm) of 50 % hydroxyalkoxypropyl Sephadex (prepared as described by Ellingboe et aL7) packed in methanol-water (75 : 25, v/v). The I 6-unsaturated C, 9 steroids were eluted between 45 and 95 ml of effluent. The monophsulate fraction was solvolyzed and purified as described earlier’. The rb-unsaturated C,a steroids were separated from other more polar steroids using a 200 -mg silicic acid column packed in ethyl acetate-toluene (I 2: 88, v/v). The effluent collected between I and 4 ml contained the t6-unsaturated C,, steroids. Before analysis by gas-liquid chromatography the steroids were converted to their trimethylsilyl ether or U-methyloxime derivatives*. The analyses were performed on 3 o/1QF-I and 2.2 7: SE-30 columns. Gas chromatography-mass spectrometry was performed using a computerized (Varian, Spectra System IOO MS) combined gas
16-ANDROSTENES
IN HUMAN
TESTIS
253
chromatograph-mass spectrometer (Varian, Model CH 7). The energy of the bombarding electrons was 70 eV and the ionizing current 300 PA. For quantification stigmasterol (I 0-20 pg) was added as an internal standard prior to gas-liquid chromatography. RESULTS
The same three t6-unsaturated C,, steroids were identified in both the free and monosulphate fractions. The relative retention times of the trimethylsilyl and Omethyloxime derivatives of the compounds isolated and of the corresponding reference steroids are shown in Table II. The trimethylsilyl steroid ether first eluted from both the SE-30 and QF-r columns had the same relative retention times and mass spectrum as the corresponding derivative of reference gee-androst-r6-en-3c-ol (Fig. I). It was identified, therefore, as 5@-androst-r6-en-3a-o1. A second 16-unsaturated C,, steroid isolated from the TABLE
11
RELATIVE RETENTION TIMES OF TRIMETHYLSILYL AND O-METHYLOXIME RIVATIVES OF STEROIDS IDENTIFIED IN HUMAN TESTIS TISSUE AND THOSE THE CORRESPONDING DERIVATIVES OF REFERENCE COMPOUNDS Conditions 3% QF-I, 185°C and trimethylsilyl; MO, O-methyloxime. Identificution
z.zO(, SE-30.
180°C.
Cholestane
QF-I
TMS,
SE-30
Compound from testis
Reference compound
TMS 5cc-Androst-r6-en-3x-01 5, I 6-Androstadien_3/Gol gee-Androst-16-en-38-01 ga-Androst-k6-en-3-one*
= 1.00. Abbreviations:
DEOF
Reference compound
MO
TMS
MO
TMS
MO
TMS
0.173 0.213 0.236 _
~ ~ 0.31; 0.32 0.38;
0.120
_
0.121
0.162 0.163
~ -
0.163 0.164
-
_
0.159;
_
0.163 o. I 84
0.173
-
0.212
~
0.236
-
4, I 6-Androstadien-j-one*
Compound from testis
_
MO
0.39 * Steroid
not found
in
testis
tissue.
M-(90+15) 241
Fig. I. Mass spectrum human testis.
of the trimethylsilyl
ether
derivative
of 5a-androst-r6-en-3x-01
isolated
from
254
A. RUOKONEN
Fig. 2. Mass spectrum human testis.
of the trimethylsilyl
ether derivative
of 5,J6-androstadien-3/Gol
isolated
from
testis tissue had the same relative retention times and mass spectrum as the corresponding derivative of 5, I 6-androstadien-3/I-01 (Fig. 2). Similarly, a third steroid found in the testis tissue was identified as 5c+androst- I 6-en-3/i-ol. The mass spectrum of the trimethylsilyl ether of this steroid has been published elsewhere (Ruokonen et u/.~). The concentrations of the compounds identified are given in Table III. Of the unconjugated steroids 5a-androst- I 6-en-j/3-ol was present in highest concentration, 5,16-androstadien-~/I-o1 was the next most abundant but only trace amounts of 5~ androst- I 6-en-32-01 were detected. No ketonic I 6-unsaturated C, 9 steroids were detected in this fraction. In the monosulphate fraction 5, I 6-androstadien-j/Go1 was the main I 6-unsaturated C,9 component followed by 5a-androst- I 6-en-@-ol. Here again only a trace amount of 5@-androst- r6-en-3cc-ol was detected (Table I I I). DISCUSSION
It is well known that 16-androstenes occupy an important position in steroid metabolism in boar testis. In this tissue free and sulphate-conjugated 5u-androst-r6en-3cr-ol and 5z-androst- I 6-en-J/?-o1 are synthesized from pregnenolone and subsequently secreted into the blood stream”. Furthermore, 5, t6-androstadien-@ol is TABLE
III
CONCENTRATIONS OF FREE AND C,q STEROIDS IN HUMAN TESTIS
SULPHATE-CONJUGATED TISSUE
16-UNSATURATED
The values are expressed as Dug of the free steroid in IOO g of testis tissue and are uncorrected for methodological losses. Abbreviations: F, free steroid fraction; M. steroid monosulphate fraction. Subjects
COlYlpOl~ttd
B. T.
L.R.
L. M.
F M
1.0 0.6
0.7 0.6
0.5
5. ~6-Androstadien-3B-ol
F M
10.6 8.6
I 1.8 I 2.7
18.0 18.5
5x-Androst-J6-en-38-01
F M
37.1 4.5
16.1 3.8
33.9 17.4
5x-Androst-J
6-en-3a-o1
0.6
16-ANDROSTENES
IN
HUMAN
TESTIS
255
present in boar testis”. This study demonstrates the presence of these r6-androstenes also in human testis. However, there is a marked difference in the concentrations of these compounds in the testicular tissue from these two species. In boar testis r6-unsaturated steroids are quantitatively the most important C,, steroids (Ruokonen, A. and Vihko, R., unpublished). Whereas, in human testis the 16-unsaturated steroids are present in concentrations lower than those of many of the other C,, steroids (see Ruokonen et ~1.~‘). In human urine, 5’y-androst- I6-en-jcr-01, 5/I-androst- I 6-en-ju-ol and 5, I 6androstadien-Q-01 have been detected, gx-androst- r6-en-jz-ol being the main I 6In testis tissue, this steroid was the 16-anunsaturated C,, steroid metabolite2.13. drostene present in lowest concentrations. This might indicate its rapid secretion from the testis or the fact that other sites are more important in its generation. A very interesting finding is the relatively high concentration of ga-androst-I6‘en-j/I-o1 in human testis tissue. This compound has not previously been found in man. It would seem therefore that either the secretion of this compound from the testis is very slight or that it is further metabolized before excretion by the organism. Another possibility is that a route of elimination other than urine might be preferred for the excretion of this steroid. S/I-Androst-l6-en-33-01 or the ketonic r6-androstenes were not detected in testis tissue in this study. If present, their concentrations must have been lower than 0.5 pg per IOO g tissue, the lowest level of detection possible in this study. The present results were obtained by analysing cadaver testes. When a number of free and sulphated steroids were determined in cadaver testes and in testes ectomized because of prostatic cancer”, their steroid composition was very similar. Therefore, it would seem that the present results might reflect the composition of 16-androstenes also in normal testis, but definite proof of this is lacking at present.
ACKNOWLEDGEMENTS
The author wishes to thank professor R. Vihko for his valuable criticism during this work. The skilful technical assistance of Mrs Salme Ollikainen is gratefully acknowledged.
REFERENCES I Prelong, V. and Ruzicka, L. (1944) Helv. Chim. Acta 27, 61-66 Brooksbank, B. W. L. and Haslewood, G. A. D. (1950) Biochem. J. 47, 36-43 3 Brooksbank, B. W. L. (1962) J. Endocrinol. 24, 435-444 4 Cleveland. W. W. and Savard, K. (1964) J. C/in. Endocrinol. Metab. 24, 983-987 5 Bicknell, D. C. and Cower, D. B. (1971) J. Endocrinol. 49, I-ii 6 Laatikainen, T. and Vihko, R. (1969) Eur. J. Biochem. IO, 165-171 7 Ellingboe, J., Nystram, E. and Sjiivall, J. (1970) J. Lipid Res. II, 266-273 8 Vihko, R. (1966) Acta Endocrinol. 52, suppl. 109, 1-67 9 Ruokonen, A., Vihko, R. and Niemi, M. (1973) FEBS Lerr., 31, 321-323 10 Saat, Y. A., Cower, D. B., Harrison, F. A. and Heap, R. B. (1972) Biochem. J. 129, 657-663 I I Booth, W. D. (1970) J. Reprod. Fert. 23, 533-534 12 Ruokonen, A., Laatikainen, T., Laitinen, E. A. and Vihko, R. (1972) Biochemistry II, 1411-1416 13 Brooksbank, B. W. L. and Cower, D. B. (1970) Acta Endocrinol. (Copenhagen) 63, 79-90 2
et Biophysics Acta, Scientific Publishing
316 (1973) Company,
251-255
Amsterdam
- Printed
in The Netherlands
BBA 56302 FREE
AND
IN HUMAN
AIM0
SULPHATE-CONJUGATED TESTIS
16-UNSATURATED
C,,
STEROIDS
TISSUE
RUOKONEN
Steroid Research Laboratory, Department University of Helsinki, SF-001 70 Helsinki (Received
March
26th,
ofMedico1 Chemistry, I 7 (Finland)
1973)
SUMMARY
Free and monosulphated 5a-androst- I 6-en-3x-01, 5a-androst- I 6-en-3P-ol and 5, I 6-androstadien-3/?-ol were identified and quantified in human cadaver testis tissue. Free and monosulphated steroid fractions were isolated by Sephadex LH-20 chromatography. Further separation and purification of the r6-unsaturated C,, steroids was achieved using silicic acid and hydroxyalkoxypropyl Sephadex chromatography. The steroids were finally identified by gas-liquid chromatography and gas chromatography-mass spectrometry. In the free steroid fraction the relative abundance of the r6-unsaturated C,, steroids was 5cc-androst-r 6-en-3j%ol > 5, I 6-androstadien-3/Gol > 5a-androst- I 6-en -3~01 and in the monosulphate fraction 5, I 6-androstadien-3fl-ol > 5z-androst- I 6-en-3/Gol > 5x-androst- I 6-en-3c+ol.
INTRODUCTION
5a-Androst- I 6-en-3cc-ol and 5a-androst- I 6-en-3p-ol were first discovered in boar testis tissue’. Later, Brooksbank and Haslewood” isolated 5a-androst-r 6-en-3a01 from the glucuronide fraction of human urine. Its urinary excretion is almost three times as great in men as in women 3. Adrenocorticotropic hormone (ACTH)4 administration increases urinary 5u-androst- I 6-en-3r-ol excretion in both sexes and human chorionic gonadotrophin3 administration has the same effect in normal men and in patients with the testicular feminization syndrome 5. Thus, it seems likely that both the testes and the adrenals are capable of secreting 5a-androst-16-en-3cc-ol or its precursors. The purpose of this study was to investigate the possible role of human testis in the production of 16-unsaturated C,9 steroids. Trivial and systematic 3/J-hydroxy-5-pregnen-2o-one; corticotropic hormone.
names: testosterone, 178-hydroxy-4-androsten-3-one; stigmasterol, (2@)-24-ethyl-5,2z-cholestadien-3~-ol;
pregnenolone, ACTH, adreno-
A. RUOKONEN
252 TABLE DATA Subject
I ON SUBJECTS STUDIED ~____ __~ Age Time interval (years i after death (h)
Weight of combined testes
Cause of death
(gl B.T. L.R. M.L.
24 29 41
30 5 ‘4
34.5 48.0 22.2
Cerebral
hemorrhage
cord Cerebral hemorrhage Rupture
of spinal
EXPERIMENTAL Materials
Testes were obtained from cadavers (3 subjects). Prior to analysis the testicular capsule was removed and the tissue sample weighed and subsequently stored at - 20 “C. Data on the samples are given in Table I. R@rence compounds 5x-Androst-r6-en-3a-ol was obtained from professor W. Klyne (Steroid Reference Collection, London, England). 5, rb-Androstadien-3fi-ol and 4, r6-androstadien-3-one were kindly supplied by Dr D. B. Gower (London, England). 5cr-Androst16-en-3/?-ol and 5cc-androst-r6-en-3-one were purchased from Ikapharm (RamatGan, Israel). Methods Both testes were combined for steroid determination. The tissue sample was carefully homogenized in acetone-ethanol (I : I, v/v) and chloroform-methanol (I : I, v/v) and the combined filtrates were evaporated. The residue was chromatographed on a 20-g Sephadex LH-20 column, and the free and monosulphate-conjugated steroid fractions were collected as described by Laatikainen and Vihko6. The free steroid fraction was dried in vacua and partitioned between ethyl acetate and 0.01 M NaOH. The ethyl acetate phase was dried with MgSO,, evaporated and dissolved in 2- and I -ml portions of I 2 “/, ethyl acetate in toluene and applied to a 5-g silicic acid column (0.8 cm x 18. I cm) packed in toluene. The i6-unsaturated C,, steroids were eluted from the column with 40 ml of 12 % ethyl acetate in toluene. The final purification was carried out on a 2.5-g column (0.6 cm x 18.4 cm) of 50 % hydroxyalkoxypropyl Sephadex (prepared as described by Ellingboe et aL7) packed in methanol-water (75 : 25, v/v). The I 6-unsaturated C, 9 steroids were eluted between 45 and 95 ml of effluent. The monophsulate fraction was solvolyzed and purified as described earlier’. The rb-unsaturated C,a steroids were separated from other more polar steroids using a 200 -mg silicic acid column packed in ethyl acetate-toluene (I 2: 88, v/v). The effluent collected between I and 4 ml contained the t6-unsaturated C,, steroids. Before analysis by gas-liquid chromatography the steroids were converted to their trimethylsilyl ether or U-methyloxime derivatives*. The analyses were performed on 3 o/1QF-I and 2.2 7: SE-30 columns. Gas chromatography-mass spectrometry was performed using a computerized (Varian, Spectra System IOO MS) combined gas
16-ANDROSTENES
IN HUMAN
TESTIS
253
chromatograph-mass spectrometer (Varian, Model CH 7). The energy of the bombarding electrons was 70 eV and the ionizing current 300 PA. For quantification stigmasterol (I 0-20 pg) was added as an internal standard prior to gas-liquid chromatography. RESULTS
The same three t6-unsaturated C,, steroids were identified in both the free and monosulphate fractions. The relative retention times of the trimethylsilyl and Omethyloxime derivatives of the compounds isolated and of the corresponding reference steroids are shown in Table II. The trimethylsilyl steroid ether first eluted from both the SE-30 and QF-r columns had the same relative retention times and mass spectrum as the corresponding derivative of reference gee-androst-r6-en-3c-ol (Fig. I). It was identified, therefore, as 5@-androst-r6-en-3a-o1. A second 16-unsaturated C,, steroid isolated from the TABLE
11
RELATIVE RETENTION TIMES OF TRIMETHYLSILYL AND O-METHYLOXIME RIVATIVES OF STEROIDS IDENTIFIED IN HUMAN TESTIS TISSUE AND THOSE THE CORRESPONDING DERIVATIVES OF REFERENCE COMPOUNDS Conditions 3% QF-I, 185°C and trimethylsilyl; MO, O-methyloxime. Identificution
z.zO(, SE-30.
180°C.
Cholestane
QF-I
TMS,
SE-30
Compound from testis
Reference compound
TMS 5cc-Androst-r6-en-3x-01 5, I 6-Androstadien_3/Gol gee-Androst-16-en-38-01 ga-Androst-k6-en-3-one*
= 1.00. Abbreviations:
DEOF
Reference compound
MO
TMS
MO
TMS
MO
TMS
0.173 0.213 0.236 _
~ ~ 0.31; 0.32 0.38;
0.120
_
0.121
0.162 0.163
~ -
0.163 0.164
-
_
0.159;
_
0.163 o. I 84
0.173
-
0.212
~
0.236
-
4, I 6-Androstadien-j-one*
Compound from testis
_
MO
0.39 * Steroid
not found
in
testis
tissue.
M-(90+15) 241
Fig. I. Mass spectrum human testis.
of the trimethylsilyl
ether
derivative
of 5a-androst-r6-en-3x-01
isolated
from
254
A. RUOKONEN
Fig. 2. Mass spectrum human testis.
of the trimethylsilyl
ether derivative
of 5,J6-androstadien-3/Gol
isolated
from
testis tissue had the same relative retention times and mass spectrum as the corresponding derivative of 5, I 6-androstadien-3/I-01 (Fig. 2). Similarly, a third steroid found in the testis tissue was identified as 5c+androst- I 6-en-3/i-ol. The mass spectrum of the trimethylsilyl ether of this steroid has been published elsewhere (Ruokonen et u/.~). The concentrations of the compounds identified are given in Table III. Of the unconjugated steroids 5a-androst- I 6-en-j/3-ol was present in highest concentration, 5,16-androstadien-~/I-o1 was the next most abundant but only trace amounts of 5~ androst- I 6-en-32-01 were detected. No ketonic I 6-unsaturated C, 9 steroids were detected in this fraction. In the monosulphate fraction 5, I 6-androstadien-j/Go1 was the main I 6-unsaturated C,9 component followed by 5a-androst- I 6-en-@-ol. Here again only a trace amount of 5@-androst- r6-en-3cc-ol was detected (Table I I I). DISCUSSION
It is well known that 16-androstenes occupy an important position in steroid metabolism in boar testis. In this tissue free and sulphate-conjugated 5u-androst-r6en-3cr-ol and 5z-androst- I 6-en-J/?-o1 are synthesized from pregnenolone and subsequently secreted into the blood stream”. Furthermore, 5, t6-androstadien-@ol is TABLE
III
CONCENTRATIONS OF FREE AND C,q STEROIDS IN HUMAN TESTIS
SULPHATE-CONJUGATED TISSUE
16-UNSATURATED
The values are expressed as Dug of the free steroid in IOO g of testis tissue and are uncorrected for methodological losses. Abbreviations: F, free steroid fraction; M. steroid monosulphate fraction. Subjects
COlYlpOl~ttd
B. T.
L.R.
L. M.
F M
1.0 0.6
0.7 0.6
0.5
5. ~6-Androstadien-3B-ol
F M
10.6 8.6
I 1.8 I 2.7
18.0 18.5
5x-Androst-J6-en-38-01
F M
37.1 4.5
16.1 3.8
33.9 17.4
5x-Androst-J
6-en-3a-o1
0.6
16-ANDROSTENES
IN
HUMAN
TESTIS
255
present in boar testis”. This study demonstrates the presence of these r6-androstenes also in human testis. However, there is a marked difference in the concentrations of these compounds in the testicular tissue from these two species. In boar testis r6-unsaturated steroids are quantitatively the most important C,, steroids (Ruokonen, A. and Vihko, R., unpublished). Whereas, in human testis the 16-unsaturated steroids are present in concentrations lower than those of many of the other C,, steroids (see Ruokonen et ~1.~‘). In human urine, 5’y-androst- I6-en-jcr-01, 5/I-androst- I 6-en-ju-ol and 5, I 6androstadien-Q-01 have been detected, gx-androst- r6-en-jz-ol being the main I 6In testis tissue, this steroid was the 16-anunsaturated C,, steroid metabolite2.13. drostene present in lowest concentrations. This might indicate its rapid secretion from the testis or the fact that other sites are more important in its generation. A very interesting finding is the relatively high concentration of ga-androst-I6‘en-j/I-o1 in human testis tissue. This compound has not previously been found in man. It would seem therefore that either the secretion of this compound from the testis is very slight or that it is further metabolized before excretion by the organism. Another possibility is that a route of elimination other than urine might be preferred for the excretion of this steroid. S/I-Androst-l6-en-33-01 or the ketonic r6-androstenes were not detected in testis tissue in this study. If present, their concentrations must have been lower than 0.5 pg per IOO g tissue, the lowest level of detection possible in this study. The present results were obtained by analysing cadaver testes. When a number of free and sulphated steroids were determined in cadaver testes and in testes ectomized because of prostatic cancer”, their steroid composition was very similar. Therefore, it would seem that the present results might reflect the composition of 16-androstenes also in normal testis, but definite proof of this is lacking at present.
ACKNOWLEDGEMENTS
The author wishes to thank professor R. Vihko for his valuable criticism during this work. The skilful technical assistance of Mrs Salme Ollikainen is gratefully acknowledged.
REFERENCES I Prelong, V. and Ruzicka, L. (1944) Helv. Chim. Acta 27, 61-66 Brooksbank, B. W. L. and Haslewood, G. A. D. (1950) Biochem. J. 47, 36-43 3 Brooksbank, B. W. L. (1962) J. Endocrinol. 24, 435-444 4 Cleveland. W. W. and Savard, K. (1964) J. C/in. Endocrinol. Metab. 24, 983-987 5 Bicknell, D. C. and Cower, D. B. (1971) J. Endocrinol. 49, I-ii 6 Laatikainen, T. and Vihko, R. (1969) Eur. J. Biochem. IO, 165-171 7 Ellingboe, J., Nystram, E. and Sjiivall, J. (1970) J. Lipid Res. II, 266-273 8 Vihko, R. (1966) Acta Endocrinol. 52, suppl. 109, 1-67 9 Ruokonen, A., Vihko, R. and Niemi, M. (1973) FEBS Lerr., 31, 321-323 10 Saat, Y. A., Cower, D. B., Harrison, F. A. and Heap, R. B. (1972) Biochem. J. 129, 657-663 I I Booth, W. D. (1970) J. Reprod. Fert. 23, 533-534 12 Ruokonen, A., Laatikainen, T., Laitinen, E. A. and Vihko, R. (1972) Biochemistry II, 1411-1416 13 Brooksbank, B. W. L. and Cower, D. B. (1970) Acta Endocrinol. (Copenhagen) 63, 79-90 2