Camp. Biochem. Physiol. Vol. 97A. No. 2, pp. 115-l 17, 1990 0300-9629/90 $3.00 + 0.00 Printed in Great Britain 0 I990 Pergamon Press plc

OCCURRENCE OF MAMMALIAN SEX STEROIDS IN THE FREE-LIVING NEMATODE, TURBATRIX AC&‘7

HUI-MINN LEE,* EDWARD J. PARISH* and LEON W. BONES

*Department of Chemistry, Auburn University, AL 36849, USA. Telephone: (205) 844-4043; and tRegiona1 Parasite Research Laboratory, ARS, USDA, PO Box 952, Auburn, AL 36830, USA

(Received 12 January 1990)

Abstract-l. Mammalian sex steroids and cholesterol were isolated from the lipid extract of the free-living nematode, Turbatrix aceti.

2. The mammalian steroid sex hormones, progesterone, testosterone and estrone, were detected and isolated from the mixture.

3. The steroids were analysed by thin-layer, gas-liquid and high-performance liquid chromatography and their structures confirmed by proton nuclear magnetic resonance and mass spectroscopy.

INTRODUCTION

The evolution of endocrine systems has been exten- sively investigated (Sandor and Mehdi, 1979). These studies indicate the existence of proteins that bind mammalian sex steroids in a number of primitive species, including fungi and unicellular eukaryotes (Feldman et al., 1984). Other studies have demon- strated the existence of mammalian sex steroids in yeast and higher plants (Hassan and Wafa, 1947; Bennett et al., 1966; Kuanta, 1968; El Amin, 1969; Heftmann, 1975; Saden-Krehula et al., 1971, 1976, 1983).

These authors have recently described the presence of cytosolic binding proteins and/or receptors for sex steroids in the zooparasitic nematodes Tricho- strongylus colubriformis (Kiser et al., 1986) and Nippostrongylus brasiliensis (Majumdar et al., 1987). Additional studies have established the pres- ence of the mammalian sex steroids, progesterone, testosterone, estrone and estriol, in T. colubri- formis and have shown that incubation with tritiated progesterone resulted in the formation of 17c(- hydroxyprogesterone (Chung et al., 1986; Lee et al., 1989).

In these studies of the steroid content of nematodes, the search for additional species that may contain sex steroids has been extended. In the present communication the aim was to show that the free- living nematode. Turbatrix aceti, contains the sex steroids progesterone, testosterone and estrone.

MATERlALS AND METHODS

The free-living nematode, T. aceti, was incubated for 14 days in a medium (Bolla, 1977) containing haemoglobin, sitosterol, cholesterol and ergosterol. Nematodes were washed with distilled water, filtered, and homogenized with 2: 1 chloroform-methanol (v/v) to a final 1:20 dilu- tion (w/v). The aqueous phase was reextracted with chloroform-methanol and deried with 0.02% magnesium chloride (Folch et al.. 1957: Kuksis. 1977). After sol- vent evaporation, residues were dissolved’ in a small amount of methanol and eluted through a reverse phase Sep-Pak cartridge (Waters Associates) with water and

methanol as mobile phases (Shackleton and Whitney, 1980). The solution was concentrated under nitrogen at 45°C and dried in a vacuum desiccator. The residue was dissolved in 2ml of absolute ethanol and steroids in this solution were analysed by thin-layer chromatog- raphy (TLC), gas-liquid chromatography (GLC) and high- performance liquid chromatography (HPLC). The dry weight of worms was determined by taking 10% of their wet volume and drying at 90°C prior to weighing the sample.

GLC analysis was conducted using a Shimadzu (GC-9 A) gas chromatograph, equipped with a flame ionization detec- tor and a Chromatop AC computer operating at a tem- perature gradient of 220°C (1 min) to 290°C (4’Cjmin). Glass columns (6 ft), packed with Gas-Chrom Q (100/200 mesh), containing 2% SE-30 (Chitwood er al., 1983, 1985), were used with a nitrogen flow rate of 20ml/min. Values reported (Table I) are based on GLC analysis of the lipid extent using internal standards (five analyses were con- ducted) (Kuksis, 1977; Tomsova et al., 1981; Lee et al.. 1989). Conditions for TLC and HPLC analysis have been described (Chung et al., 1986). TLC solvent systems were: ether-toluene (35: 65). hexane-ether (50: 50) and chloroform-ether (25 : 75).

Steroids were isolated [for melting point (MP), mass spectrometry (MS) and proton nuclear magnetic resonance (PMR) analysis] on TLC plates (20 x 20 cm silica gel G glass plates (Analtech, Newark, DE)] which were activated at 105°C for 40 min (Freeman and West, 1966; Renwick et al., 1983). Plates were eluted with hexane-ether (1: 1) and steroids visualized by iodine vapour (Torchstone, 1986), removed, extracted with chloroform-methanol (2: 1). evap- orated, and dried prior to analysis. Conditions for MP determinations, MS and PMR analysis have been described (Parish et al., 1987).

Authentic standards of cholesterol [after purification (Fieser, 1953)], progesterone, testosterone and estrone were obtained from Sigma Chemical Co. (St Louis. MO).

RESULTS AND DISCUSSION

Several reports have described the physiological effects of mammalian sex steroids on nematodes. Testosterone and diethylstilbesterol influenced the fecundity of Trichinella spiralis (Reddington et al., 1981), while testosterone was found to increase the recovery of N. brasiliensis from mice and cause a

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116 H.-M. LEE et al.

Table 1. Steroid content of Turbafrix acefi

Steroid Wt% in dry worm Wt% in lipid

Cholesterol 0.275% + 0.033% 0.825% * 0.099% Progesterone 0.0176% + 0.0033% 0.0528% + 0.010% Testosterone 0.0297% + 0.0024% 0.0891% f 0.0073% Estrone 0.0357% f 0.0056% 0.113% f 0.017%

Values reported are from GLC analysis of the lipid extracts using internal standards.

dose-dependent production of eggs (Swanson et al., 1984). In addition, progesterone was found at pico- gram per milligram levels in the free-living nematode, Panagrellus redivivus (Willett, 1980). Recently, these authors have reported the presence of sex steroid receptors for progesterone, testosterone, and 17fl- estradiol in an extract from the ruminant nematode, T. colubriformis (Kiser et al., 1986). These results led to the isolation of progesterone, testosterone, estrone, and estriol from the same organism (Chung et al., 1986; Lee et al., 1989). In companion studies, these authors have demonstrated steroid receptors for pro- gesterone, testosterone and 17fl-estradiol in an ex- tract from the rodent hookworm, N. brasiliensis (Majumdar et al., 1987). These latter results have led to the development of new steroid antihelminthic agents using sex steroids as models (Majumdar et al., 1987).

In the present study, the occurrence of the mam- malian sex steroids, progesterone, testosterone and estrone was shown in the lipid extract from the free-living nematode T. aceti (Table 1). This is the first report describing the presence of a complement sex steroids in a free-living nematode species. In addition, cholesterol was isolated and identified as the major steroid present in this organism. These steroids had identical chromatographic properties upon TLC, GLC and HPLC analysis with authentic standards. The steroids described in Table 1 were isolated by TLC and subjected to PMR and MS analysis. The spectra of each isolated steroid were identical in all respects to those obtained from the respective standard compounds. An analysis of the

Table 2. Proton nuclear magnetic resonance (PMR) and mass spectral (MS) data of steroids isolated from Turbarrix aceta

MS (relative intensity

Steroid PMR of major ions)

Cholesterol 0.80 (C-18 CH,). 386 (loo%), 368 (19%) 1.01 (C-19 CH,). 353 (9%). 326 (4%) 2.50 (C-3 H) 301 (18%). 275 (22%) 5.37 (C-6 H. vinyl) 255 (10%)

Progesterone 0.70 (C-18, CH,). 314 (91%). 272 (79%), I.21 (C-19 CH,). 244 (34%). 229 (58%). 2.13 (C-21 CH,), 191 (28%). 173 (15%). 5.70 (C-4 H. vinyl) 191 (28%). 173 (15%).

147 (30%). 124 (100%)

Testosterone 0.81 (C-18 CH,). 288 (28%). 273 (5%), 1.21 (C-19 CH,). 246 (43%). 228 (14%), 3.62 (C-17 H), 203 (19%), 165 (13%). 5.69 (C-4 H. vinyl) 147 (34%). 124 (100%)

Estrone 0.91 (C-18 CH,), 270 (100%). 213 (19%), 4.67 (C-3 OH), 18s (28%). 172 (21%). 6.56 (C-4 H, aromatic). 160 (13%). 140 (17%) 6.60 (C-2 H, aromatic), 7.12 (C-l H, aromatic)

CDCI, served as solvent and tetramethylsilane was an internal standard.

spectra obtained from each steroid isolated from T. colwbriformis is presented in Table 2. These results are consistent in all respects with the published spectra (Stenhagen et al., 1974). Finally, each steroid had a MP similar to the authentic standards, which corresponded to literature values (Windholz et al., 1983).

As a group, helminths appear unable to accomplish the biosynthesis of steroids de nova and thus require an exogenous source of steroid(s) for growth and development (Barrett, 1981). Studies have revealed that in T. aceti the major metabolic block in the isopentenoid pathway to sterol biosynthesis occurs between farnesol and squalene (Lu et al., 1977). Experiments with supplemented sterols have indi- cated that T. aceti can reproduce in a media contain- ing a variety of animal and plant sterols (Cole and Dutky, 1969; Lu et al., 1977) and possess the ability to convert phytosterols to cholesterol via C-24 dealkylation (Cole and Krusberg, 1968). In addition, a novel nuclear C-4 sterol alkylation pathway has also been found (Chitwood et al., 1987).

The finding of a complement of mammalian sex steroids in this free-living organism indicates that the evolved enzymatic system necessary for androgen and estrogen biosynthesis may be more prevalent among lower animals than believed previously. Also, contin- ued studies using T. aceti as a model system may lead to a better understanding of the function and mech- anism of steroid hormone action in a variety of organisms.

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