Fish Physiology and Biochemistryvol. 6 no. 2 pp 91-112 (1989) Kugler Publications, Amsterdam/Berkeley

Steroids and steroid glucuronides in the ovarian fluid of the African catfish, Clarias gariepinus, between ovulation and oviposition W . G . E . J . Schoonen, J.C.M. Granneman and J.G.D. Lambert Department o f Experimental Zoology, Research Group o f Comparative Endocrinology, University of Utrecht, Padualaan 8, 3584 CH Utrecht, The Netherlands Keywords: gas chromatography-mass spectrometry, steroids, steroid glucuronides, ovarian fluid, pheromones

Abstract

As part of a series of experiments concerning a possible pheromonal function of steroids and steroid glucuronides excreted by the sex organs of the African catfish, Clarias gariepinus, qualitative and quantitative studies, using GCMS, were carried out to examine the presence of the steroids, that can be synthesized by the ovary during oocyte maturation and ovulation, and of the corresponding steroid glucuronides, in the fluid surrounding the eggs in the ovarian cavity shortly after ovulation. Full mass spectra were obtained of 5/3-pregnane-3a, 17u-diol-20-one, 5~-pregnane-3a,17~,20~-triol, 58pregnane-3a,6c~,17ct-triol-20-one, 5B-pregnane-3a,6a,17u,20/3-tetrol, 5/3-androstane-3a,1713-diol and 5/3androstane-3t~, 17/3-diol-1 l-one. After selected ion monitoring the following steroids could be detected by the presence of at least two characteristic ions at the expected retention time: 5~-pregnane-3t~,17c~,20~-triol, etiocholanolone, 5~-dihydrotestosterone, 5~-androstane-3c~, 1 l~-diol-17-one, testosterone and estradiol. After treatment with/3-glucuronidase the following steroids could be determined in a similar way: 5/3-pregnane3ct, 17ff-diol-20-one, 5/3-pregnane-3ct, 17ct,20~-triol, 5B-pregnane-3ct, 17ct,20/3-triol, 5~-pregnane-3c~,6t~, 17~triol-20-one, 5B-pregnane,3u,6c~, 17u,20B-tetrol, 5/3-androstane-3t~, 17B-diol, etiocholanolone, 5/3-dihydrotestosterone, testosterone and estradiol. The free steroids 58-pregnane-3a,6c~,17~,208-tetrol and 58-pregnane-3a,6ct,17a-triol-20-one and the steroid glucuronides of testosterone, 58-dihydrotestosterone and estradiol aplbeared to be the most abundant o f these compounds. The results indicate that very polar steroids and steroid glucuronides, synthesized in the ovary, can be excreted via the ovarian fluid shortly before and during oviposition, and possibly function as sex attractants, inducing reproductive behaviour in male conspecifics.

Introduction

Field studies have shown that in early summer, after ovulation, female African catfish enter the inundated shore of the lake in which they live; males follow the track of these females. As soon as a male

reaches a female, spawning takes place (Bruton 1979; Lambert et al. 1986). It is likely that spawning behaviour will be influenced by pheromones. There is some evidence that in males steroid glucuronides synthesized in the seminal vesicle can act as sex attracting substances (Resink et al. 1987a, 1987b;

Correspondence to: J.G.D. Lambert, Department of Experimental Zoology. Research Group of Comparative Endocrinology, University of Utrecht, Padualaan 8, 3584 CH Utrecht, The Netherlands

92 Schoonen and Lambert 1987). Reciprocally, in the field and under laboratory conditions following ovulation, female African catfish appear to attract male conspecifics (Resink, personal communication). This attraction of males by ovulated females might be induced by ovarian pheromones. Indeed, in other species of teleosts, ovarian pheromones can elicit male reproductive behaviour (Van den Hurk et al. 1982; Van den Hurk and Lambert 1983; Liley and Stacey 1983), and there are indications that these pheromones are steroids and/or steroid glucuronides (Colombo et al. 1982; Van den Hurk and Lambert 1983; Johanson 1985). If that is also the case in African catfish these compounds would have to be present among the steroids and steroid glucuronides in the fluid surrounding the eggs in the ovarian cavity. As a first step in the study of the nature of ovarian sex pheromonbs in Clarias gariepinus, the steroids and steroid glucuronides were identified and their concentration was measured in the ovarian fluid of adult African catfish following induced ovulation and stripping. In vitro experiments have shown that during ovulation the ovary of the African catfish can synthesize fourteen free steroids and four steroid glucuronides (Schoonen et al. 1988a). These fourteen steroids and their glucuronides were examined and their concentration was measured by means of gas chromatography-mass spectrometry in the fluid surrounding the eggs shortly after induced ovulation. In addition, following preliminary results, the ovarian fluid was analyzed for the presence of 5/3-androstane-3c~, 17~-diol-11-one, 5/3-androstane-3u, 1l~-diol- 17one, cholesterol and cholesterol- glucuronide; the concentration of the first two of these compounds was measured.

(Netherlands) photoperiod and fed with trout pellets. Four mature animals, 14 months old weighing 750 g were used for the experiments. Ovulation was induced by intraperitoneal injection of pimozide and LHRHa, 5/zg and 0.05 #g/g body weight, respectively, suspended in 0.8~ NaC1 solution with 0.1 ~ sodium metabisulphite and 0.25~ bovine serum albumin (De Leeuw et al. 1985). Sixteen hours after injection, i.e. approximately 2 - 4 h after ovulation, the animals were anesthetized with 2~ phenoxy-ethanol. The ovaries were removed, and eggs and ovarian fluid were collected by stripping the ovaries. The eggs and ovarian fluid, as well as the remaining ovaries were weighed; the average weight being 86.15 _+ 4.82 and 25.0 + 1.17 (mean _+SEM) g/egg mass and the rest of the ovary, respectively. A 40g sample of the eggs and ovarian fluid of each fish was centrifuged at 3000 rpm for 10 min. The supernatant, i.e. the ovarian fluid, was collected. The eggs were washed twice with distilled water and recentrifuged in order to collecting as much of the ovarian fluid as possible.

Chemicals

All chemicals and solvents were of analytical grade and the solvents (Baker) were distilled twice before use. Reference steroids were obtained from Steraloids and Makor, [6,9(n)3Hlestrone-3/~-D-glucuronide (sp. act. 19.6 Ci/mmol) from Radiochemical Centre, Amersham, B-glucuronidase of Escherichia coli (100 U/ml) from Boehringer, Seppak C18columns from Waters Associates, trimethylsilylimidazole (TSIM) from Supelco, Pennsylvania, U.S.A., pimozide from Janssen Pharmaceuticals Ltd, Beerse, Belgium and LHRH-analogue (desGly-[D-ala6]LHRH-ethylamide) from Intervet International B.V., Boxmeer, The Netherlands.

Materials and methods Animals

Extraction

Female African catfish, C. gariepinus, were reared in the laboratory according to De Leeuw et al. (1985). The fish were kept in a copper free circulation system at 25 +_ 2~ exposed to a natural

Free steroids and steroid conjugates were extracted from the ovarian fluid by reversed-phase chromatography with Seppak Cls-columns. These columns were activated with methanol (2 x 2 ml) and equili-

93 brated with distilled water (2 • 5 ml). After this pretreatment ovarian fluid of each of the four experimental animals was diluted with distilled water to a volume of 10 ml and transferred to a column. After rinsing the column with 10 ml distilled water to remove remaining proteins, both steroids and steroid conjugates were eluted with ethanol (4 x 2 ml) and ethanol-distilled water (1:1) (4 x 2 ml). The eluates of each column were evaporated under a stream of nitrogen and the residue redissolved in distilled water (5 ml) to dissolve the steroids and steroid conjugates. Thereafter the free steroids were extracted with dichloromethane (3 • 10 ml) and this free steroid fraction was set apart until derivatization. In order to eliminate contamination of the steroid conjugate fraction with free steroids, the remaining water fraction was washed again with dichloromethane ( 3 x 1 0 ml) and subsequently evaporated. The remaining water residue was redissolved in 2 ml sodium acetate buffer (0.1 M, pH 6.5) and treated with I00 ~1 /3-glucuronidase at 37~ overnight under continuous shaking in an atmosphere of air. The enzyme reaction was terminated by the addition of 10 ml dichloromethane, whereafter extraction of deglucuronidated steroids was carried out with dichloromethane. To control the efficiency o f the extraction 0.4 ~Ci [3H]estrone-glucuronide, dissolved in 100 ~1 50~ aqueous ethanol, and 60 ng 5-pregnene-3~,17u, 20~-triol, dissolved in 100 #1 propylene glycol, were added as internal standards to the ovarian fluid of each sample. This method could be adopted, because preliminary experiments had shown that the efficiency of extraction of several steroid glucuronides corresponded with the efficiency o f extraction of estrone-glucuronide, and that of steroids with that o f 5-pregnane-3f3,17~,20f3-triol, all being approximately 85 to 90~ Moreover, this glucuronide and free steroid can not be synthesized by ovaries under in vitro conditions (Schoonen et al. 1988a). For these reasons Seppak Cl8-columns were used for the purification of steroid glucuronides from ovarian fluid. The suitability of this procedure for separating free steroids has been reported by Shackleton and Whitney (1980) and Heikkinen et al. (1981).

Derivat&ation Derivatization towards trimethylsilyl (TMS) and oxime-trimethylsilyl-steroid-derivatives was carried out as follows: dichloromethane fractions with free or deglucuronidated steroids were transferred to reaction vials. During the process of derivatization 60 ng 5-pregnene-3/3, i 7u,20/3-triol was added as an internal standard to the vials containing deglucuronidated steroids. Because of the extraction procedure, this c o m p o u n d was already present in the vials containing free steroids, and therefore was not added again. Then, the vials were dried under a stream of nitrogen, whereafter 200/~1 2~ hydroxyla m m o n i u m chloride in pyridine was added. These mixtures were incubated for one hour at 100~ Under these conditions, steroids possessing keto groups were converted to oxime derivatives. Following evaporation, 100 ~1 TSIM was added to the residue and incubated for 2 h at 150~ to obtain the TMS ether derivatives of the steroids (Sakauchi and Horning 1971). After evaporation, the residue was dissolved in 2 ml hexane and the polar compounds (non-steroid-derivatives) were removed by extraction with acetonitrile ( 2 x 0 . 2 ml). Finally, the steroid trimethylsilyl and oxime-trimethylsilyl derivatives were dissolved in 12 p.l hexane and an aliquot of 2 p.l was subjected to GC-MS.

Capillary gas chromatography-mass spectrometry A Hewlett-Packard 5992 B gas chromatograph-, mass spectrometer with a H . P . fused silica capillary column (Ultra 1, cross-linked methyl silicone, film thickness, 0.17 #, 25 m x 0.21 m m id) was used with helium as carrier gas at a flow rate of 2 m l / min. The injection port temperature was 250~ and the oven temperature was set at 160~ and was made to increase 1 min after injection at a rate of 15~ up to 190~ H a l f a minute after this temperature was reached, the temperature was made to increase further with 2 ~ to 235~ For total ion monitoring with a scan reach of 2 0 0 660 m / z , the multiplier detector was set at 1800 V and for selected ion monitoring (SIM) at 2600 V. The mass spectrometer was optimalized for the mid

94 range area ( m / z 414) and the obtained mass Spectra were non-normalized spectra.

Table I. Characteristic ions of derivatized steroids and their lower detection levels with GC-MS.

Derivative of steroid

Ion Lower detection fragment level GC-MS (ng/2 #1)

17~t-Hydroxyprogesterone 17~,20B-Dihyd roxy-4-pregnen-3-one 5B-Pregnane-3m, 17c~-diol-20-one 5B-Pregnane-3t~, 17m,20~-triol 5B-Pregnane-3t~, 17ct,20B-triol 5B-Pregnane-3m,6m, 17~-t riol-20-one 5B-Pregnane-3m,6a, 17a,20B-tetrol Androstenedione Testosterone Estrone Estradiol Etiocholanolone 5B-Dihydrotestosterone 5/3-Androstane-3a,l 7B-diol 5B-Androstane-3a, 17B-diol- 1l-one 5B-Androstane-3cc, 1IB-diol- 17-one

487.4 446.3 422.2 255.0 255.0 510.3 523.2 460.0 447.2 340.0 416.2 270.0 449.2 256.0 360.0 448.2

Spectra comparison-similarity index Both the spectra of the steroids derived from the ovarian fluid and of reference steroids were reduced to 10 peaks, selected on the base o f the highest mass times abundance values. With regard to the reference steroids, these 10 particular peaks were stored in the library. The algorithm used in comparing the reduced spectra of the unknowns from ovarian fluid with those o f the reference steroids in the library were based on the following correlation index or similarity index equation, given as a standard p r o g r a m m e in the H P - G C M S :

2.50 2.50 1.09 0.10 0.10 0.36 0.65 2.50 3.98 0.50 0.10 0.12 1.56 0.10 0.05 0.99

k -m= S.I. =

•/

k m=l

1 Am'am Am 2

k "m=l

am2

in which S.I. = similarity index (0 _< S.I. < 1) (0 = totally different, 1 = identical) A m = abundance of the ion at mass m in unknown spectrum. a m = abundance of the ion at mass m in library spectrum. k = total number of different ions (10 _< k < 20) S.I. > 0.8 was used as arbitrary value for a good correlation.

determination, are given in Table 1. In this way, linear calibration curves (0,8349 < r < 0,9708, n _> 29) were calculated after twofold measurements of a duplo analysis. With this method the lower detection level of steroids varied from 100 pg to 3,98 ng per injection (Table 1). The reliability o f these curves was c o m p a r a b l e to those shown by Schoonen et al. (1988b) and Van D a m et aL (1988). For 17a-hydroxyprogesterone, 17ct,20~-dihydroxy-4-pregnen-3-one, androstenedione, testosterone and 5/3-dihydrotestosterone relatively high detection levels were found. The relatively low sensitivity was caused by the appearance of a cis and trans configuration after derivatization, resulting in two peaks for each steroid.

Calibration curves Calibration curves of the steroids were prepared in the concentration range o f 0,2 to 30 ng per 2 #1. After derivatization of these steroids and of the internal standard (5-pregnene-3/3,17ot,20/3-triol), and after applying GC-MS, using selected ion monitoring (SIM), the ratio of the areas of one of the most characteristic ions of a particular steroid and of the characteristic ion ( m / z 252.8) of the standard, was determined (Shackleton 1986; Ichimura et al. 1986). The characteristic fragment ions, used for

Results

Separation o f steroids by gas chromatography A standard gas c h r o m a t o g r a p h y (GC) run with a mixture of equal amounts (200 ng) o f the steroid derivatives resulted in a sufficient separation of most of the c o m p o u n d s . This is shown for the compounds with a retention time between 20.0 and 33.0 min in Fig. 1. Testosterone (oxime-diTMS), 5fl-di-

95

GC-Run

Min.

Steroid derivative

20.1

5/~-Androstane-3ar, 17j~ -diol [diTMS]

22.7 23.4 23.6 24.2 24.5 24.9

Etiocholanolone (oxirne- diTMS} Estradiol {diTMS) 51~-Androstane-3or,t 7~ -diol-t 1-one (diTMS} 5fl,-Dihydrotestosterone {oxime-diTMS} {cis} 5~-Dihydrotestosterone [oxime-diTMS) (Vans) 5]~ -Androstane-3or, 17J3-diol- 1 t-one {oxime-triTMS}

26.1 27.0 27.4

Eslrone (oxime-diTMS} 5/3-Pregnane-3or, 17or,20/3-Viol (triTMS} 5~-Androstane-3or, 11~ -diol- 17-one (oxime-triTMS} Testosterone (oxlrne-diTMS} (Vans}

28.3 28 8

5,B-Pregnane-30:,17of ,20dr-triol (triTMg} 5~-Pregnane-3or,17or -diol-20-one (oxirne-triTMS}

22

23L~'~ 24" c 25"

E

26-

c

Co 27

29.9 30.4

a3

If-

32.5 32.8

)

Androstenedione (dioxime-diTMS}(cis} Androstenedione (dioxime-diTMS)(trans} S/~,-Pregnane-3~,6aL 17ot,20~ -felt ol (tetraTMS}

5~ -Pregnane-3oL6o~,17a~-triol- 20-one (oxime-tetraTMS) 5-Pregnene-3~, 17o~,20~ -triol {lriTMS}

Fig. 1. Capillary gas chromatogram of derivatives of standard steroids (200 ng).

hydrotestosterone (oxime-diTMS), androstenedione (dioxime-diTMS), 17~-hydroxyprogesterone (dioxime-triTMS) and 17~,20B-dihydroxy-4-pregnen-3-one (oxime-triTMS) all manifested themselves in a cis and trans configuration, which resulted in two peaks of each steroid. The cis configuration o f testosterone (oxime-diTMS) co-chromatographed with the derivatives o f 5~-pregnane3et, 17c~,20B-triol and 5B-androstane- 3~, 1 l/3-diol17-one and the trans configuration of androstenedione (dioxime-diTMS) co-chromatographed with the derivative o f 5~-pregnane-3u,6c~, 17c~,20B-tetrol. This, however, did not interfere with the identification of these steroids as their derivatives have their own characteristic mass fragments. The derivative of 5B-androstane-3c~,17B-diol-ll-one also showed two peaks, with retention times (RT) of 23.6 and 24.9 min, respectively. This indicates the presence of two different derivatives, i.e. 5B-androstane-3t~,17B-diol-ll-one (diTMS) with a MW of 450.2 and 5B-androstane-3u,17~-diol-ll-one (oxime-triTMS) with a MW of 537.4. As the retention time o f 17u-hydroxyprogesterone (dioxime-triTMS) (RT cis = 42.8 min, RT trans = 46.0 min), 17u,20/3-dihydroxy-4-pregnen-

3-one (oxime-triTMS) (RT cis = 38.8 min, RT trans = 41.3 min) and cholesterol (TMS) (RT = 43.0) was more than 33.0 min, these compounds are not shown in Fig. 1.

Identification by GC-MS 5B-Androstane-3ct,1713-diol: The TMS derivative of standard 5B-androstane-3~, 17B-diol had a retention time of 20.1 mih. The mass spectrum was characterized by the molecular ion with m / z 436.2 (M+), and the mass fragment ions'with m / z 421.2 (M § , m / z 346.0 (M § and m / z 256.0 (M+-2 x OTMS) (Fig. 2A). The abundance ratio for the ions 436.2, 346.0 and 256.0 was 15:48:100, respectively. Total ion monitoring for the free steroid fraction of the ovarian fluid resulted in a full spectrum at 20.2 min (Fig. 2B), which demonstrated the presence of 5B-androstane-3~, 17B-dioldiTMS, since this spectrum correlated very well (S.I. = 0.979) with the mass spectrum of the standard. SIM analysis of the molecular ion m / z 436.2 and the fragment ions with m / z 346.0 and m / z 256.0 showed that these ions were present at a reten-

96 A

5,/3-Androstane-3ce, 17/3-diol CdiTMS) 256.0 M§

346.0

M+-90 J

l

J. !+.:250IL.,..., 300

....

421.2 M+-15

,..,.

200

. . . . . .

350

436.2 M+

t.,, 450

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400

B

Ovarian

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500

i

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550

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fluid Steroid (free)

.. k ,, ,,J 300 ,

200

250

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350

,

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400

,

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Ions

F S . 2 1 ~

436.2

i

,

,

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+

,

550

.

,

600

steroid [glucuronide) i FSm544

436.2 ._.~.

~

_-,',d~.....-.....;,.,,_..,;,

FSm544

F5=2175

346.0

346.0

FS~544

FS=~2175

256.0

,

500

Steroid (free) ions

9

450

i [

256.0

,.'!

.,

,

,

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r

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20 25 30 A Retention time {min.)

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.

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Retention time (min)

Fig. 2. A. Mass spectrum (non-normalized) of 5/~-androstane-3c~,17/~-diol-diTMS standard. The characteristic ions are the molecular ion m / z 436.2 and the mass fragment ions m / z 421.2, m / z 346.0 and m / z 256.0 B. Mass spectrum (non-normalized) of the derivatized free steroid fraction of the ovarian fluid of C. gariepinus at the expected retention time of 5/~-androstane-3c~,17/~-diol-diTMS. C. SIM analysis of the derivatized steroid and steroid glucuronide fractions of the ovarian fluid of C. gariepinus between 19 and 33 rain of the GC-run. The characteristic ions of 5/3-androstane-3a, 17/~-diol-diTMS were present at the retention time of 20.2 min in both fractions.

tion time of 20.2 rain in both the free steroid and steroid glucuronide fractions with abundance ratios of 14:46:100 and 15:45:100, respectively (Fig. 2C). Etiocholanolone: The retention time of standard

etiocholanolone-oxime-diTMS was 22.7 min. Its characteristic ions were the molecular ion with m/z 449.2 (M § and the mass fragments with m/z 434.2 (M § m/z 360.0 (M § and m/z 270.0 (M§ x OTMS) (Fig. 3A). The abundance

97

A

Etiocholanolone 270.0 M+.179

Coxime-diTMS)

360~0 M+-90

I

JL

,J. . I"

200

'

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250

.

'

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300

.

.

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350

B

.

.

434.2 449,2 M +- 15 M+

~. L,

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400

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.

.

.

.

.

.

.

I

450

''

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9

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9

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600

fluid

Steroid (free)

Ions

'

'

500

Steroid (glucuronide) Ions

FS=651

449.2

FS--

136

449.2

i;! L', .

~H

~. . . .

!i !i!

Fs=,3~

FS-651

j

360.0

360.0

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;::

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270.0

I S

270.0 .

! . . . . i

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R e t e n t i o n time (rain]

I

20

i

i

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A

=

I

25

i

=

,

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310

Retention time (min)

Fig. 3.A. Mass spectrum (non-normalized) of etiocholanolone-oxime-diTMS standard. The characteristic ions are the molecular ion m / z 449.2, and the mass fragment ions m / z 434.2, m / z 360.0 and m / z 270.0. B. SIM analysis of the derivatized steroid and steroid glucuronide fractions of the ovarian fluid of C. gariepinus between 19 and 33 min of the GC-run. The characteristic ions of etiocholanolone-oxime-diTMS were present at te retention time of 22.7 rain in botb fractions.

ratio of the ions 449.2, 360.0 and 270.0 was 6:45:100. Total ion monitoring did not result in a full spectrum. On the other hand, SIM analysis demonstrated that the fragment ions with m/z 449.2, m/z 360.0 and m/z 270.0 were found at a retention time of 22.7 min in the free steroid and steroid glucuronide fractions (Fig. 3B). The abundance ratios appeared to be 14:55:100 for the free steroid fraction and 8:45:100 for the steroid glucuronide fraction.

Estradiol-17fl: The mass spectrum o f the diTMS derivative o f estradiol-17/3 was characterized by the molecular ion with m / z 416.2 (M +) and the mass fragment with m / z 285.1. The retention time o f this c o m p o u n d was 23.4 min (Fig. 4A). The abundance ratio for the ions 416.2 and 285.1 was 100:97 and the theoretical abundance ratio for M + (416.2), M + + 1 (417.2) and M + + 2 (418.2) was 100:37:14. After analysis o f the free steroid and steroid glucuronide fractions, it appeared that in both fractions estradiol could be identified with SIM analy-

98

A

Estradiol- 1 7~ (diTMS) 285.1 M+-131

.I ~

!

9

,

,

9

401.2

M+-90

..,

200

M+

326.2

I .~

tu~.JA.J

416.2

i, ,

Jt

i

,

,

.

250

,

i

.

M+-15

I[ .

.

300

t .

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9

350

B

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450

400

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1 84 ,

i

9

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550

500

!

600

Ovarian fluid S t e r o i d (free) ions

Steroid [glucuronide) Ions

FS~'281

417.2

!

i

Fs-zel

FS=161

417.2

.

FS=161

416.2

i FS=281

285.1

]

416.2

i :

,:

FS = 161

i :.::

:,*, ,

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::.,

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,L

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~.,~ , ' : 20

i

285.1

,-; ,=,', A 25

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30

Retention time (min}

i

1

20

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& 25 Retention

i

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30 time (min)

Fig. 4.A. Mass spectrum (non-normalized) of estradiol-17/~-diTMS standard. The characteristic ions are the molecular ion m / z 416.2, and the mass fragment ions m / z 401.2, m / z 326.2 and m / z 285.1. B. SIM analysis of the derivatized steroid and steroid glucuronide fractions of the ovarian fluid of C. gariepinus between 19 and 33 min of the GC-run. The characteristic ions of estradiol-17/~-diTMS were present at the retention time of 23.4 min in both fractions.

sis, only. The SIM runs of three ions are shown in Fig. 4B. From these runs the abundance ratio was calculated for the ions 416.2 vs 285.1 (100:75) and of M § M§ and M § (100:39:11) for the free steroid fraction, and of 416.2 vs 285.1 (100:79) and of M § M § + 1 and M § + 2 (100:39:16) for the steroid glucuronide fraction.

5fl-Androstane-3o~,l 7fl-diol-l I-one:

From this standard two derivatives were found: 5/~androstane-3u,17fl-diol-I 1-one-diTMS with a retention time o f 23.6 min and a mass spectrum with

a molecular ion with m / z 450.2 (M § and mass fragments with m / z 435.2 (M+-CH3), m / z 360.0 (M § -OTMS), m / z 345.2 (M § -(OTMS + CH3)) and m / z 306.1 (M § • TMS) (Fig. 5A I). The abundance ratio of the ions 450.2, 360.0 and 306.1 is 26:100:90. The second derivative, 5flandrostane-3o~, 17/~-diol- 11- one-oxime-triTMS had a retention time of 24.9 min and a mass spectrum with the following typical ions: molecular ion with m / z 537.4 (M § and mass fragments with m / z 522.4 (M+-CH3), m / z 446.3 (M§ m/z 432.3 ( M + - ( O T M S + C H 3 ) ) and m / z 358.2 (M +-

99 2• The abundance ratio for the ions 537.4, 446.3 and 358.2 was 87:100:81 (Fig. 5A II). Both derivatives could be identified in the derivatized free steroid fraction of the ovarian fluid by spectral analysis (Fig. 5B I and II). The spectra correlated very well with the standard spectra (S.I.: 0.978 and 0.923). With SIM analysis the characteristic ions of 5/3androstane-3c~,17~-diol-diTMS, i.e. m / z 450.2, m / z 360.0 and m / z 306.1 could be detected in the free steroid ovarian fluid fraction at a retention time of 23.6 min (Fig. 5C I) and with an abundance ratio of 30:100:91. The typical fragment ions of the second derivative, 5~-androstane-3u,17~-diol-llone-oxime-triTMS, i.e. m / z 537.4, m / z 446.3 and m / z 358.2 could also be detected in this fraction at a retention time of 24.9 min with an abundance ratio of 88:100:74 (Fig. 5C II).

5~-Dihydrotestosterone: The oxime-diTMS derivatives of 5~-dihydrotestosterone, with a cis and trans configuration, had retention times of 24.2 and 24.5 min, respectively. The mass spectra of these derivatives demonstrated the same characteristic molecular ion m / z 449.2 (M +) and mass fragments m / z 434.2 (M + -CH3), m / z 360.0 (M + -OTMS) and m / z 270.0 (M +-2 x OTMS) as etiocholanolone oximediTMS (Fig. 6A). The abundance ratio, however, appeared to be different, i.e. 100:72:41 for the ions 449.2:360.0:270.0, respectively. SIM analysis of the ions with m / z 449.2, m / z 360.0 and m / z 270.0 showed that in both ovarian fluid fractions these ions were present at a retention time of 24.2 and 24.5 min for the cis and trans configuration derivatives. The abundance ratio for these ions was 100:64:36 in the free steroid fraction and 100:71:41 in the steroid glucuronide fraction (Fig. 6B).

5~3-Androstane-3ct,11~3-diol-17-one:

The oximetriTMS derivative of 5~-androstane-3~,ll~-diol17-one had a retention time of 26.9 min and its characteristic ions were a molecular ion with m / z 537.4 (M+), m / z 358.2 ( M + - 2 • and m / z 267.8 (M +-3 x OTMS) with an abundance ratio of 9:100:22:72, respectively (Fig. 7A). With SIM analysis only the free steroid fraction of ovarian fluid

showed characteristic ions at a retention time of 27.0 min in an expected ratio of 10:100:27:67 (Fig. 7B).

513-Pregnane-3o~, 17et,20~-triol: The triTMS derivative of 5f3-pregnane-3a,17c~,20f3-triol had a retention time of 27.1 min, which was comparable to that of 5~-androstane-3c~,ll/3-diol-17-one-oximetriTMS. The mass spectrum was characterized by the molecular ion m / z 552.1 and the mass fragments with m / z 435.3 (M+-(C20 ' 21 side chain + OTMS)), m / z 345.3 (M +-(C20 ' 2J side chain + 2 x OTMS)) and m / z 255.0 (M+-(C20, 21 side chain + 3 • OTMS)) (Fig. 8A I). The abundance ratio of the ions 435.3, 345.3 and 255.0 is 43:17:100. After analysis with SIM of both the free steroids and steroid glucuronide fractions, the characteristic ions were demonstrated in both fractions at a retention time of 27.0 min (Fig. 8C). Calculation of the abundance ratios of the ions 435.3,345.3 and 255.0 resulted in a ratio for the free steroid fraction of 66:18:100 and in the steroid glucuronide fraction of 47:17:100.

Testosterone: Only the trans configuration of testosterone-oxime-diTMS with a retention time of 27.4 min was used for identification. The spectrum of the standard showed three characteristic ions, i.e. the molecular ion M + with m / z 447.2 and its fragments with m / z 432.0 (M+-CH3) and m / z 211.0 (M +-236) (Fig. 9A). The abundance ratio of 447.2 vs 432.0 was 100:46, and the theoretical ratio of the molecular isotopes m / z 447.2 (M+), m / z 448.2 ( M + + I ) and m / z 449.2 ( M + + 2 ) wa~ 100:38:14. SIM runs of the free steroid and steroid glucuronide fractions of ovarian fluid resulted in the identification of testosterone in both fractions at the expected retention time (Fig. 9B). From these runs the abundance ratio of M + and M +-CH 3 and M +, M + + I and M + + 2 appeared to be 100:46 and 100:39:14 respectively for the free steroid fraction and 100:48 and 100:39:15 respectively for the steroid glucuronide fraction. 5{3-Pregnane-3ct, 17c~,20c~-triol: The triTMS derivative of 5/3-pregnane-3a,17~,20c~-triol showed a re-

]00 A

I

5~-Androstane-3~, 17/~-diol- 1 1-one (diTMS]

306.1 M+- 144

345.2 M*- 101

Ii ,,,1.. l.iL 200

. IL

250

450.2

i

,

,

,

350

300

I

i

,

,

L ,

I

400

,

'1

1

,

450

!

,

,

,"

'

500

I

'

,

,

r

550

I

600

5~-Androstane-3~, 17/~-diol- 1 1-one (oxime-triTMS)

11

358.2

M"10i III M.-gf I

250

300

350

B

1537.4

432.3446.3

M+" 179

200

M*

435.2 M%15

'

'

'

'

400

i

,

M'-t5522"4 |l/

,

,

450

1

,

i

,

i

500

M+

I

,

,

]

550

i

I

600

Ovarian fluid ! Steroid (free]

],Jill I1,[ H=ll,~,liJ 2~0 . . . .

=I

250 . . . .

'

I

300 . . . .

,,l

350 . . . .

,

I,

400 . . . .

J

,

,

450 . . . .

,I

500 . . . .

5~0 . . . .

600

'IT Steroid (free]

260 ....

2go ....

C

360

Z

' ' 'ago ....

460 ....

4go ....

560 ....

Steroid [free]

660

Steroid (free]

Tr

Ions

5go ....

Ions FS=340

450.2

FS=

t70

FS=

170

537.4

,~=.o I

,

360.0

J

i

i!

ii

i

446.3

. i ~,,,-,~~ ---,...~,L_,.~.~..~,~_j,_ , .:"-,---~-~:J .,,

!.

,._

,).

L

~cS= 340

FS=

306,1

170

I

358.2

.

'2'o'

' 'A' 2'5 ....

3'0'

Retention time (min)

' '

.

.

'2'0 ....

.

J

4

t

9

~5

3'0' ' '

Retention time (rnirO

101

A

5/3-Dihydrotestosterone 360.0 M+-90

434.2 M+-15

270.0 M+-179

|

I

=

[.

li d

=

'

200

lJl

I'

h

l

,

250

300

~

,

r'

T

|

,

,

350

B

[oxime-diTMS]

,

I '

I I

i

449.2 M+

Jl ,

,

400

,

,

450

'

~5(~0'

'6~o

' ' '550'

Ovarian fluid Steroid Ions

[free)

Steroid [glucuronide} Ions

FS= '95

449.2

FS=204

ii

449.2

il

l:

"'

,-,..

............ FS = 195

360.0

11

!~

360.0 i

IL " ~9s Fs~

270.0

,, ~ ~---:'~

FS=204

-_:

=

.......

=_

i :::" ' ii :' ,:::', .,,,. :..~:, j ~ ' - . . ' : '-_~--., ~: -'~.,_,,,

: i'i,..~_.:| iJ\~_-~=~ _~.,-._~.,.=,,,~ ., ....,"-~

f

i

~. /'

i ~.

,I T~ , - , ' ' ; 7", .... , , , , 20 kt25 30 R e t e n t i o n time (rain)

~

FS= 204

270.0

~.~

"..

i

J

":

I

20

~t25

30

Retention time (rain)

Fig. 6.A. Mass spectrum (non-normalized) of 53-dihydrotestosterone-oxime-diTMS standard. "['he characteristic ions are the molecular ion m / z 449.2, and the mass fragment ions m / z 434.2, m / z 360.0 and m / z 270.0. B. SIM analysis of the derivatized steroid and steroid glucuronide fractions of the ovarian fluid of C. gariepinus between 19 and 33 rain of the GC-run. The characteristic ions o f 53-dihydrotestosterone-oxime-diTMS were present at the retention tithe of 24.2 (cis) and 24.5 (trans) min in both fractions.

Fig. 5.A. Mass spectra (non-normalized) of 5/3-androstane-3a,173-diol-I I-one-diTMS (I) and 53-androstane-3(x,17,6'-diol-I l-oneoxime-triTMS (II) standards. The characteristic ions are (I) the molecular ion m / z 450.2, and the mass fragment ions m / z 435.2, m / z 360.0 and m / z 306.1 and (11) the molecular ion m / z 537.4 and the mass fragment ions m / z 522.4, m / z 446.3, m / z 432.3 and m / z 358.2, respectively. B. Mass spectra (non-normalized) o f the derivatized free steroid fraction o f the ovarian fluid of C. gariepinus at the expected retention times of 53-androstane-3a, 173-dio1-1 t-one-diTMS (I) and of 5/3-androstane-3a, 173-dio1-1 l-one-oxime-triTMS (II). C. SIM analysis of the derivatized steroid fraction of the ovarian fluid of C. gariepinus between 19 and 33 min of the GC-run. The characteristic ions o f 5/3-androstane-3(x,173-diol-ll-one-diTMS (I) and 53-androstane-3cx,17B-diol-ll-one-oxime-triTMS (Ii) were present at the retention times of respectively 23.6 and 24.9 min in this fraction.

102

5/}-Androstane-3a', 1 1/3 -diol- 17-one (oxime-triTMS)

A

448.2

M+-89

267.8 M*-270

2

~

1

1

1

1

1

1

i

1

1

358.2

M§

522.4

.L 1

1

1

250

,

1

,

400

350

Ovarian fluid

B

Steroid (free) Ions

FS:

78

537.4

ii;i ii

" .

"

~.,,-.

.

~LF~

FS=391

448.2

,,.::

FS~391

358.2

i

!--

"

tI

::i

267.8

,c--: :

_

,

2'0

.

i

. '- [ - ' ,

i

,

I

2s Retention

i

--

i~

-:i~~.~,

,

L i

.,

:: n"

i\

.

. i

k

k

1

300

,

.i .-~"

. ,

|

i

30

t i m e (mJn)

Fig. 7.A. Mass spectrum (non-normalized) of 5t3-androstane3~,1 lfl-diol-17-one-oxime-triTMS standard. The characteristic ions are the molecular ion m / z 537.4, and the mass fragment ions m / z 522.4, m / z 448.2, m / z 358.2, and m / z 267.8. B. SIM analysis of the derivatized free steroid fraction of the ovarian fluid of C. gariepinus between 19 and 33 min of the GCrun. The characteristic ions of 5fl-androstane-3cqll/~diol-17-one-oxime-triTMS were present at the retention time of 26.9 min.

537.4

M+.15 M § L.k 450

'56o

.

.

.

.

5 o'

600

tention time of 28.3 min, while the characteristic ions of the mass spectrum were identical to those of 5fl-pregnane-3cql7cq20/3-triol-triTMS, i.e. m / z 552.1, m / z 435.3, m / z 345.3 and m / z 255.0 (Fig. 8A II) and had an abundance ratio of 42:10:100 for the ions 435.3, 345.3 and 255.0, respectively. The same spectrum could also be detected in the free steroid fraction of ovarian fluid with a similarity index of 0.977 and with a retention time of 28.2 min (Fig. 8B). The SIM runs of the ions with m / z 435.3, m / z 345.3 and m / z 255.0 demonstrated that besides the free steroid fraction also the steroid glucuronide fraction contained 5fl-pregnane-3c~,17cq20c~-triol (Fig. 8C). The abundance ratio for these ions was 60:13:100 for the free steroid fraction and 46:10: 100 for the steroid glucuronide fraction.

5~-Pregnane-3ee, 17o~-diol-20-one: The

oximetriTMS derivative of 5/3-pregnane-3cql7~-diol-20one had a mass spectrum with characteristic ions of m / z 565.3 (M+), m / z 550.3 (M +-CH3), m / z 476.3 (M +-OTMS), m / z 422.2 (M +-2 x TMS) and m / z 246.1 (M+-319) (Fig. 10A). The retention time of this compound was 28.8 min. The abundance ratio of the ions 565.3, 476.3, 422.2 and 246.1 is 11:69:100:73. The same spectrum could be detected in the free steroid fraction o f the ovarian fluid with a retention time of 28.8 min and a similarity index of 0.998 (Fig. 10B). In the free steroid fraction as well as in the steroid glucuronide fraction the ions 565.3,476.3,422.2 and 246.1 were demonstrated at a retention time of 28.6 min (Fig. 10C) with abundance ratios o f 12:73:100:58 and 11:69:100:52, respectively.

5/3-Pregnane-3u, 63,17u,20fl-tetrol: The tetraTMS

103 A

I 5~-Pregnane-3tz,

17o: ,20/~-triol ( t r i T M S )

255.0 M+-297

435.3 M%117 345.3 M -207 ,

!

200

.

.

.

.

I

250

I ] IL

. 9

'

'

35O

300

'

7

'

]

'

L '

400

i

~

.

552.2 M* ,

,

450

r

'

=

500

'

1

600

550

]I 5/~-Pregnane-3c~, 17o:,20c~-triol (triTMS) 255.0 M+'297

435.3 M+o117 345.3 M'-207

9 j J,

260'

1

552.1 M*

.L

' '

'250

. . . .

. . . . .

3 0 0

350'

B

. . . . 4 .0 0. . . . .

'560'

450

' '

'550'

9

' "660

Ovarian fluid H Steroid [free]

...~(,

,,.

'

C

.....

'300

,,~

. . . .

Steroid

II 350

~I~ .... . . . .

, 400'

, '

[free]

' '5~o . . . .

' '560'

'

Steroid

(glucuronide)

o6o

Ions

Ions FS-945

435.3

435.3

! i!

:

Fs = g,l$

FS:8 16

345.3

345.3

9

FS-=945

,

.

,

"!~-

.

~$=816

255.0

'2'o

.

.

.

.

i

2'5' ir l li '3b'

Retention time [min)

i . . . . .

'

'

.

!

255.0

ii

.

, 210 . . . .

,~...;~

215'

i

~'

.~....,..~

3,0 '

'

'

Retention lime (min)

Fig. 8.A. Mass spectra (non-normalized) o f 5B-pregnane-3u,17t~,20B-triol-triTMS (I) a n d 5B-pregnane-3ct, 17ct,20a-triol-triTMS (11) standards. T h e characteristic ions are the molecular ion m / z 552.1 and the mass fragment ions m / z 435.3, m / z 345.3 and m / z 255.0. B. Mass spectrum (non-normalized) o f the derivatized free steroid fraction o f the ovarian fluid o f C. gariepinus at the expected retention time of 5B-pregnane-3c~,17a,20ot-triol-triTMS. C. SIM analysis o f the derivatized steroid a n d steroid glucuronide fractions of the ovarian fluid of C. gariepinus between 19 and 33 min of the G C - r u n . The characteristic ions o f both 5fl-pregnane-3ct,17a,20/%triol-triTMS and 5fl-pregnane-3ct,17oc,20ct-triol-triTMS were present at the retention times of respectively 27.0 and 28.2 min in both fractions.

104

A

Testosterone (oxime-diTMS) IM 11.0 +-236

i

, ,tit J,i ,

,

432. i M*-15

, = . ,.~ _. ~

,

!

200

i

J

,

,

,

250

I

= "ul

~,'

,

300

k

II

,

i

350

B

i

!

i

,

,

447.2 M+

,

400

,

450

,

i

,

,

500

,

,

!

,

550

'

I

600

Ovarian fluid (free)

Steroid

Ions

FS -

Steroid

Ions

651

[glucuronide]

FS.509

448.2

448.2

FS=

~51

FS=509

447.2

447.2 ,,.,,

FS=509

FS=651

432.0

432.0 ,J i

I

'

'

i

,

20

I

25

i

~a

AA

Retention time

i

i

/

i

3O

(min)

2~0

25

AA

Retention time

30 [min)

Fig. 9.A. Mass spectrum (non-normalized) of testosterone-oxime-diTMS standard. The characteristic ions are the molecular ion m / z 447.2, and the mass fragment ions m / z 432.0 and m / z 211.0. B. SIM analysis of the derivatized steroid and steroid glucuronide fractions of the ovarian fluid of C. gariepinus between 19 and 33 min of the GC-run. The characteristic ions of testosterone-oxime-diTMS were present at the retention time of 27.1 (cis) and 27.4 (trans) min in both fractions.

derivative of 5/~-pregnane-3a,6ec,17a,20/3-tetrol had a retention time of 30.4 min. The mass spectrum showed a molecular ion with m / z 640.4 (M +) and other mass fragments with m / z 550.3 (M § OTMS), m / z 523.2 ( M + C 20, 21 side chain + OTMS)), m / z 343.2 (M § ' 21 side chain + 3 x OTMS)) and m / z 253.2 (M § ' 2] side chain + 4 • OTMS)) with an abundance ratio of 6:75:47:100 for the ions 640.4, 523.2, 343.2 and 253.2, respectively (Fig. l lA). This mass spectrum was also obtained from the free steroid fraction of the -

ovarian fluid (Fig. 11B I) with a S.I. of 0.980. SIM analysis further demonstrated that these ions were also present in the steroid glucuronide fraction. The retention time was 30.3 min and the abundance ratios were 8:88:44:100 for the free steroid fraction and 5:56:36:100 for the steroid glucuronide fraction (Fig. 11C).

'5~-Pregnane-3~, 6~, 17c=,20oL-tetrol': Although authentic material was not available, the mass spectrum of this derivatized c o m p o u n d seems to be

105 A

5~-Pregnane-3~, 17~-diol-20-one (oxime-triTMS) 246.1 M+-319

422.2 1 M+-149

,.,+~,,-4~.rJ!J~.L..:.

200

r.[,,. 300

250

:..

I

'

'

'

,L '

350

B

I

476.3 M+.89

,l .

'

'

'

'

400

:,,r

I

565.3 M*

550.3 M*-15 I

450

'

'

'

'

I

500

'

'

'

'

550

I

600

Ovarian fluid Steroid (free)

l~Jll+

lJl, I

~I I+, .... I

, ,.l t/ ,., ,

200

250

300

c

350

i

,

, I

'

i

,L ,

t ,

400

I

,

i

,

450

'

J

'

'

I,

I

'

'

'

'

550

I

600

Steroid (glucuronide) Ions

FS = 497

I

500

Steroid (free) Ions

J

26

FS=

565.3

565.3

9

"

-.-% . . . .

476.3

FS=2130

476.3

FS=

153

422.2

FS=2130

422.2

FS=

153

246.1

rs=~3o

246.1

F S =

153

~.

:',..

,.

;'~

~_.:."

ii

.....

2'5

A 'o

Retention time (min)

210

!

i

i

!

T 251

i

i

, ~'

301

'

.

i

Retention time (min)

Fig. 10. A. Mass spectrum (non-normalized) of 5B-pregnane-3u, 17u-diol-20-one-oxime-triTMS standard. The characteristic ions are the molecular ion m / z 565.3 and the mass fragment ions m / z 550.3, m / z 476.3, m / z 422.2 and m / z 246.1. B. Mass spectrum (non-normalized) of the derivatized free steroid fraction of the ovarian fluid o f C. gariepinus at the expected retention time of 5B-pregnane-3ct,17t~-diol-20-one-oxime-triTMS. C. SIM analysis of the derivatized steroid and steroid glucuronide fractions o f the ovarian fluid of C. gariepinus between 19 and 33 min o f the G C - r u n . The characteristic ions o f 5B-pregnane-3t~, 17a-diol-20-one-oxime-triTMS were present at the retention time of 28.6 min in both fractions.

106

A

I 5/~-Pregnane-3~',6c~,

17o~ ,20,~-tetrol

(tetraTMS)

253.0

523.2

H+-387

343.2

M *- 117

L _|,.,L.J,L ..... t 200

550.3

L . . . . . .

250

300

350

400

B

640,4 M+

l, M+,"90

, 450

500

550

600

650

Ovarian fluid

I Steroid

(tree) RT 30.4 min

~j,. JikJ, W, ,....'~"}"". 300 200 250 r

,

,

,

I

'

'

'

350

400

'

'

'

450

500

550

'

I

600

650

I/ Steroid [tree)

o

.

.

.

.

I, .

.

1

.

.

2

.

Ib iI

,

,

300

C

,

RT

,,, J

.

.

.

350

.

,,J I i

400

,

i

,

, .

.

.

.

.

.

.

450

,

,

500

i

,

,

i

550

Steroid [free)

.

.

.

.

32.0 min

J

.

.

.

.

i

600

650

Steroid [glucuronide)

Ions

Ions FS=

118

FS=

640.4

19

640.4

:

l

I*

FS=1060

523.2

523.2

343.2

343.2 :: FS=

5,

1060

F S i T ~

253.0

253.0 .~j,~.~.j..-,JL:'..,-v%.j

'2'0

.... 2'5 .... Retention time

v~ :',j 9 L j

3'6i

(mini

'.~

"~'

'2b .... 2'5 .... J~ ~: "~r' Retention time (rain]

Fig. II.A. Mass spectrum (non-normalized) of 53-pregnane-3a,6~,17a,20B-tetrol-tetraTMS standard. T h e characteristic ions are the molecular ion m / z 640.4 and the mass fragment ions m / z 523.2, m / z 342.2 and m / z 253.0. B. Mass spectra (non-normalized) of the derivatized free steroid fraction o f the ovarian fluid of C. gariepinus at the expected retention time o f 53-pregnane-3a,6~,17a,203-tetrol-tetra TMS and at the retention time o f 32.0 min.

107 c o m p a r a b l e with that of 53-pregnane-3a,6c~,17ct, 203-tetrol-tetraTMS, just as the mass spectra of the derivatives of 53-pregnane-3et,17et,20/3-trioltriTMS and 53-pregnane-3a,17a,20c~-triol-triTMS resembled each other. Spectrum analysis at a retention time of 32.0 min resulted in a spectrum identical to that of 53-pregnane-3a,6a,17et,20/3-tetroltetraTMS (Fig. 11B II), suggesting the presence of a 53-pregnane-3a,6a, 17a,20a-tetrol-tetraTMS. SIM analysis of both the free steroid and steroid glucuronide fraction demonstrated the presence of the characteristic ions m / z 640.4, m / z 523.2, m / z 343.2 and m / z 253.2 at a retention time of 31.9 min. The abundance ratio for the free steroid and steroid glucuronide fractions appeared to be 3:55:32:100 and 0:43:33:100, respectively.

53-Pregnane-3a,6a, 17a-triol-20-one: The oximetetraTMS derivative of 53-pregnane-3c~,17atriol-20-one showed a retention time of 32.5 min. The mass spectrum of this steroid gave rise to a molecular ion with m / z 653.4 and to the characteristic ions with m / z 564.3, m / z 510.3 and m / z 246.1 (Fig. 12A) with an abundance ratio of 15:74:92:100, respectively. After derivatization of the steroid and steroid glucuronide fractions of the ovarian fluid, a mass spectrum could be obtained of the free steroid fraction at a retention time of 32.7 min (Fig. 12B) with a similarity index of 0.940. SIM analysis showed that the characteristic ions were present both in the free steroid and steroid glucuronide fractions at a retention time of 32.6 min with abundance ratios of 16:75:91:100 and 11:38:49:100, respectively (Fig. 12C).

Cholesterol: Cholesterol-TMS could also be demonstrated in both the free steroid and steroid glucuronide fractions. A full spectrum o f cholesterol-TMS could be detected at a retention time of 43.0 min. (Fig. 13B). The mass spectra correlated very well with those of the standard (Fig. 13A). The

similarity index was 0.983 for the free steroid fraction and 0.964 for the steroid glucuronide fraction.

Other steroids: In addition to the steroids mentioned above, also the presence of estrone, androstenedione, 17c~-dihydroxyprogesterone and 17ct, 203-dihydroxy-4-pregnen-3-one was investigated in the free steroid and steroid glucuronide fractions. After derivatization of the ovarian fluid fractions these four steroids could, however, not be demonstrated by spectral analysis. Even SIM analysis on the characteristic ions did not lead to the identification of these compounds, in one of the ovarian fluid fractions of six animals, used in this or a pilot experiment.

Quantification o f the identified steroids by means o f SIM Quantification o f the derivatized free steroids and steroid glucuronides, identified in ovarian fluid, resulted in the data described in Table 2. As far as the free steroids are concerned, high levels were found of 53-pregnane-3c~,6a,17c~-triol-20-one and 53-pregnane-3a,6cq 17a,203-tetrol; intermediate levels of 53-pregnane-3a, 17c~-diol-20-one, 53-pregnane-3a, 17c~,20a-triol, 53-pregnane-3c~,6cq 17c~, 20c~-tetrol, testosterone, etiocholanolone, 53-dihydrotestosterone, 53-androstane-3u,173-diol and 53-androstane-3a,113-diol-17-one, and very low levels of 53-preg.nane-3a,17a,203-triol, estradiol and 53-androstane-3u,173-diol-ll-one. With regard to the steroid glucuronides, the levels of testosterone and 53-dihydrotestosterone were highest, while the levels of 53-pregnane-3a,6a,17c~-triol20-one, 53-pregnane-3cq6a,17u,20u-tetrol, 53pregnane-3a, 6a,17~,203-tetrol, estradiol, etiocholanolone and 53-androstane-3a,173-diol were intermediate. 53-pregnane-3a,17c~-diol-20-one, 53pregnane-3a, 17u,2Ou-triol and 53-pregnane-3a, 17c~, 203-triol were only found at very low levels,

C. SIM analysis of the derivatized steroid and steroid glucuronide fractions of the ovarian fluid of C. gariepinusbetween 19 and 33 min of the GC-run. The characteristic ions of 53-pregnane-3a,6ct.17a,203-tetrol-tetraTMS were present at the retention time of 30.3 min in both fractions, but also at the retention time of 31.9 rain.

108

A

5/3-Pregnane-3o~,6o~,17or-triol-20-one 246.1 M+-407

i

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.

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(oxime-tetraTMS] 564.3 M+-90

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653.4 M*

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'l jLL,

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Steroid (free)

r t .,

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Steroid (glucuronide)

Ions

Ions FS=590

FS=32

653.4

564.3

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653.4

FS ~2982

FS=367

564.3

FS=2982

FS=367

510.3

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246. I

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Retention time (min]

Fig. 12.A. Mass spectrum (non-normalized) o f 5B-pregnane-3c(,6(~, 17o~-triol-20-one-oxime-tetraTMS standard. The characteristic ions are the molecular ion m / z 653.4 and the mass fragment ions m / z 564.3, m / z 510.3 and m / z 246.1. B. Mass spectrum (non-normalized) of the derivatized free steroid fraction o f the ovarian fluid o f C. gariepinus at the expected retention time of 5B-pregnane-3c~,6a,17c(-triol-20-one-oxime-tetraTMS. C. SIM analysis of the derivatized steroid and steroid glucuronide fractions of the ovarian fluid of C. gariepinus between 19 and 33 min of the GC-run. The characteristic ions of 5B-pregnane-3cx,6ot,17o(-triol-20-one-oxime-tetraTMS were present at the retention time of 32.6 min in both fractions.

109 A

Cholesterol 329.3 M+-129

(TMS)

368.3 458.3 M+ 443.3 M+-15

t l

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Fig. 13.A. Mass spectrum (non-normalized) of cholesterol-TMS standard. The characteristic ions are the molecular ion m / z 458.3 and the mass fragment ions m / z 443.2, m / z 368.3 and m / z 329.3. B. Mass spectra (non-normalized) of the derivatized steroid and steroid glucuronide fractions of the ovarian fluid of C. gariepinus at the expected retention time of cholesterol-TMS

and the ll-oxygenated steroids were completely absent in this fraction.

Discussion In vitro inculgation of ovarian tissue of the African catfish, C. ~ga a r iepinus, sho w ed that at the time of

oocyte maturation and ovulation tfitiated pregnenolone and androstenedione can be converted into a number of steroids and steroid glucuronides. Among the steroids produced by these tissue fragments were 53-pregnane-3u,17~-diol-20-one, 53pregnane-3o~,17~,20~-triol, 53-pregnane-3~,17~,-

203-triol, 53-pregnane-3ct,6c~, 17t~-triol-20-one, 53pregnane-3ct,6o~,17~,203-tetrol, t~stosterone, estradiol, etiocholanolone and 53-androstane-3ct,173-diol (Schoonen et al. 1988a). In the present experiments these compounds were also found among the steroids in the ovarian fluid surrounding the eggs shortly after ovulation. The same holds for the glucuronides of 5B-pregnane-3~,17t~- diol-20-one, testosterone, 5B-dihydrotestosterone and 5B-androstane-3t~, 173-diol. It seems, therefore, that in vitro incubation studies of minced steroid producing tissues with tritiated precursors, apart from reflecting the activity of steroidogenic enzyme systems, provide

110 Table 2. Levels of steroids and steroid glucuronides in ovarian fluid of African catfish, C. gariepinus, in vivo. Steroid

17u-Hydroxyprogesterone 17u,20B-Dihydroxy-4-pregnen-3-one 5/3-Pregnane-3c~, 17u-diol-20-one 5B-Pregnane-3a, 17u,2Oot-triol 5B-Pregnane-3a, 17c~,20B-triol 5B-Pregnane-3c~,6u, 17u-triol-20-one 5B-Pregnane-3u,6ct, 17t~,20u-tetrol* 5B-Pregnane-3u,6u, 17t~,2OB-tet rol Androstenedione Testosterone Estrone Estradiol Etiocholanolone 5B-Dihydrotestosterone 5/3-Androstane-3a, 17~-diol 5/3-Androstane-3c~, 17/~-diol- 11 -one 5B-Androstane-3ot, 1 lB-diol- 17-one

Free

Glucuronide

(ng/ovary)

(ng/ovary)

ND ND 66.8+32.6 62.3 + 47.7 7.7+_ 2.9 429.2+62.8 125.6_+26.5 366.2_+37.6 ND 183.1 _+49.9 ND 12..7_+ 1.5 112.4_+26.8 81.3 _+29.8 68.1 _+ 12.1 21.9+_ 6.9 54.0_+32.1

ND ND 31.1_+ 2.6 8.8 + 0.8 9.5_+ 1.8 76.0+ 4.8 73.6_+ 12.5 56.0_+ 3.2 ND 264.4_+51.1 ND 98.3+_ 9.7 49.8_+ 2.6 242.9 _+39.8 95.6_+ 10.5 ND ND

Data are shown as means _+ SEM (n = 4); ND = not detectable; * quantified with the calibration curve of 5/3-pregnane-3a,6a, 17c~, 200tetrol.

some information as to the metabolic end products to be secreted, in this case by the ovaries into the ovarian fluid. It should be emphasized, however, that certain steroids such as 17c~-hydroxy-progesterone, 17ct,20/3-dihydroxy-4-pregnen-3-one, androstenedione and estrone, albeit present in the circulation (Van Dam et al. 1988), could not be found in the ovarian fluid. The variety of steroids and especially steroid glucuronides, detected in the ovarian fluid was greater than in the incubation experiments. There were strong indications for the presence of 5/3pregnane-3a,6a,17c~,20a-tetrol, and also of 5/3dihydrotestosterone and cholesterol. Not only the above mentioned glucuronides, but also the glucuronides of 5/3-pregnane-3a, 17c~,20a-triol, 5/3pregnane-3a, 17c~,20~-triol, 5/3-pregnane-3~,6c~, 17c~-triol-20-one, most likely 5/3-pregnane-3c~,6a,17a,20a-tetrol, 5/3-pregnane-3a,6ot, 17a,20/3-tetrol, estradiol, etiocholanolone and cholesterol were found in the ovarian fluid. Obviously, the capacity of the ovaries for steroid glucuronidation was more pronounced than appeared from the results of the incubation experiments. The ovarian fluid also contained 5~-androstane-3a,17/3-diol-ll-one and 5/3-

androstane-3c~,l l~-diol-17-one. These ll-oxygenated steroids were not demonstrated among the metabolites of steroid precursors in in vitro incubation experiments (Schoonen et al. 1988a). The relatively high concentrations of 5/3-pregnane-3c~,6et, 17a-triol-20-one, 5~-pregnane- 3c~, 6c~,17t~,20/3tetrol, testosterone-glucuronide and 5~-dihydrotestosterone-glucuronide are in line with the results of the in vitro incubation experiments (Schoonen et al. 1988a). In these experiments, however, a considerable production of 5~-pregnane- 3et,-17cr-diol-20one and 5/~-pregnane-3c~, 17c~,20/3-triol was observed, but these compounds are present in the ovarian fluid at not more than a medium or low concentration. Probably, these steroids are not only metabolic end products, but to a large extent intermediates, which are further converted into 5/3-pregnane-3c~,6c~, 17ct-triol-20-one and 5/3-pregnane-3c~, 6t~, 17c~,20~-tetrol, respectively. Furthermore, the polar steroids 5/3-pregnane3c~,6et,17~-triol-20-one and 5~-pregnane-3c~,6et,17c~,20/3-tetrol and the glucuronides of testosterone, 5/3-dihydrotestosterone and estradiol were some 2 to 10 times more abundant than other free or glucuronidated steroids. The relatively

111 high concentration of estradiol-glucuronide in the ovarian fluid does not conform the steroidogenic capacity of the ovaries in the period preceding ovulation. At that time the in vitro synthesis of estradiol is very low and estradiol-glucuronide cannot be produced at all (Schoonen et al. 1988a). It seems that following induced ovulation and stripping the ovarian fluid of the African catfish contains a large variety of steroids and steroid glucuronides. Among these may be one or more compounds that elicit spawning behaviour in male conspecifics. That would be in line with the conclusion of Columbo et al. (1982) that etiocholanoloneglucuronide attracts male goldfish and guppies. Likewise, 17~,20/3-dihydroxy-4-pregnen-3-one was found to function as a primer pheromone in the goldfish (Stacey and Sorensen 1987) and estradiol could be used as a female sex attractant in the guppy (Johansen 1985). This makes it worthwile to look for the female sex pheromone of the African catfish among the steroids and steroid glucuronides identified in the ovarian fluid surrounding the eggs after ovulation

Acknowledgements The authors thank Prof.Dr. P.G.W.J. Van Oordt for his constructive criticism on the manuscript, Mr. M.J. Van Oosterum and Mr. H.C. Schriek for taking care of keeping the animals, Ms. M.H. Van Hattum for typing the manuscript, the Image Processing and Graphic Design Department for making the figures and Dr. J.Th. Gielen, Intervet International B.V., Boxmeer, The Netherlands, for the supply of LHRHa. The investigations were supported by Research Funds of the Utrecht University.

References cited Bruton, M.N. 1979. The breeding biology and early development of Clarias gariepinus (Pisces: Clariidae) in Lake Sibaya, South Africa with a review of breeding in species of the subgenus Clarias (Clarias). Trans. Zool. Soc. Lond. 35: 1-45.

Colombo, L., Colombo Belv6d/~re, P., Marconato, A. and

Bentivegna, F. 1982. Pheromones in teleost fish. In Proceedings of the International Symposium on Reproductive Physiology of Fish, Wageningen. pp. 84-94. Edited by C.J.J. Richter and H.J.Th. Goos. Pudoc, Wageningen. De Leeuw, R., Resink, J.W., Rooyakkers, E.J.M. and Goos, H.J.Th. 1985. Pimozide modulates the luteinizing hormonereleasing hormone effect on gonadotrophin release in the African catfish, Clarias lazera. Gen. Comp. Endocrinol. 58: 120-127. Heikkinen, R., Fotsiani, T. and Adlercreutz, H. 1981. Reversedphase Ci8 cartridges for the extraction of estrogens from urine and plasma. Clin. Chem. 27: 1186-1189. lchimura, K., Yamanaka, H., Chiba, K., Shinozuka, T., Shiki, Y., Saito, K., Kusano, S., Amenya, S., Oyoma, K., Nozaki, Y. and Kato, K. 1986. Simultaneous quantitative measurement of fourteen adrenal steroids by capillary column gas chromatography-mass spectrometry, and its clinical application. J. Chromatog. 374: 5-16. Johansen, P.H. 1985. Female pheromone and the behaviour of male guppies (Poecilia reticulata) in a temperature gradient. Can. J. Zool. 63: 1211-1213. Lambert, J.G.D., Van den Hurk, R., Schoonen, W.G.E.J., Resink, J.W. and Van Oordt, P.G.W.J. 1986. Gonadal steroidogenesis and the possible role of steroid glucuronides as sex pheromones in two species of teleosts. Fish Physiol. Biochem. 2: 101-107. Liley, N.R. and Stacey, N.E. 1983. Hormones, pheromones, and reproductive behavior in fish. In Fish Physiology Volume IXB. pp. 1-63. Edited by W.S. Hoar, D.J. Randall and E.M. Donaldson. Academic Press, New York. Resink, J.W., Van den Hurk, R., Groeninx-Van Zoelen, R.F.O. and Huisman, E.A. 1987a. The seminal vesicle as source of sex attracting substances in the African catfish, Clarias gariepinus. Aquaculture 63: I 15-128. Resink, J.W., Van den Hurk, R., Peters, R.C. and Van Oordt, P.G.W.J. 1987b. Steroid glucuronides as sex attracting pheromones in the African catfish, Clarias gariepinus. Proc. Third Int. Symp. Reprod. Physiol. Fish, St. Johns, Newfoundland: 163. Sakauchi, N. and Horning, E.C. 1971. Steroid trimethylsilyl' ethers, derivative formation for compoun~ls with highly hindered hydroxyl groups. Anal. Lett. 4: 41-52. Schoonen, W.G.E.J. and Lambert, J.G.D. 1987. GC-MS analysis of steroids and steroid glucuronides in the seminal vesicle fluid of the African catfish, Clarias gariepinus. Gen. Comp. Endocrinol. 68: 375-386. Schoonen, W.G.E.J., Lambert, J.G.D., Penders, M.T., Van Roosmalen, M.E., Van den Hurk, R., Goos, H.J.Th. and Van Oordt, P.G.W.J. 1988a. Steroidogenesis during induced oocyte maturation and ovulation in the African catfish, Clarias gariepinus. Fish Physiol. Biochem. This issue. Schoonen, W.G.E.J., Lambert, J.G.D. and Van Oordt, P.G.W.J. 1988b. Quantitative analysis of steroids and steroid glucuronides in the seminal vesicle fluid of feral spawning and feral and cultivated non-spawning African catfish, Clarias gariepinus. Gen. Comp. Endocrinol. 70: 91-100.

112 Schackleton, C.H.L. 1986. Profiling steroid hormones and urinary hormones. J. Chromatog. 379: 91-156. Schackleton, C.H.L. and Whitney, J.O. 1980. Use of Seppak cartridges for urinary steroid extraction: evaluation of the method for the use prior to gas chromatographic analysis. Clin. Chim. Acta 107: 231-243@. Stacey, N.E. and Sorensen, P.W. 1987. 17ot,20fl-Dihydroxy-4pregnen-3-one: a steroidal primer pheromone increasing milt volume in the goldfish, Carassius auratus. Can. J. Zool. 64: 2412-2417. Van Dam, G.H., Schoonen, W.G.E.J., Lambert, J.G.D. and

Van Oordt, P.G.W.J. 1988. Plasma profiles of fourteen ovarian steroids before, during and after ovulation in African catfish, Clarias gariepinus, determined by gas chromatography and mass spectrometry. Fish Physiol. Biochem. In press. Van den Hurk, R., Hart, L.A. 't, Lambert, J.G.D. and Van Oordt, P.G.W.J. 1982. On the regulation of sexual behaviour of male zebrafish, Brachydanio rerio. Gen. Comp. Endocrinol. 46: 403. Van den Hurk, R. and Lambert, J.G.D. 1983. Ovarian steroid glucuronides function as sex pheromones for male zebrafish, Brachydanio rerio. Can..I. Zool. 61: 2381-2387.