The conversion of androstenedione to testosterone by some lobster (Homarus americanus Milne Edwards) tissues

GENERAL

AND

COMPARATIVE

The

ENDOCRINOLOGY

Conversion some Lobster

9,

(1967)

319-324

of Androstenedione’ (Homarus americanus

to Testosterone Milne Edwurds

Tissues Wt. W. GILGAN Fisheries

Research

Board

AKD

of Canada, Halifax Received March

Il.

R.

IDLER

Labo7atoryt

Halijax,

Noua

Scotia

17, 1967

Lobster t&is has been demonstrated to contain 17Phydroxysteroid dehpdrogenase by its capacity to reduce [4-“Cl androst-4-ene-3,17-dione to 14-“Cl testosterone. The major product of the incubation moved with carrier [1,2-“HI testosterone, during thin-layer chromatography, could subsequently be recrystallized with testoeterone with no significant change in ‘G3H ratio, and moved as the carrier [1,2-%I testosterone during high-resolution paper chromatography. The compound ~-as oxidized to another which did not separate from carrier androstenedione during paper chromatography and repeated crystallization. The products of androstenedione reduction by other lobster tissues were identified as testosterone in a similar bul, less rigorous manner. A comparison of the ability of the examined tissues to synthesize testosterone on a tissue-weight basis indicated that the androgenic gland was the most active. The vas dcferens mucosa and the testis had similar activity.

Charniaux-Cotton (1956) reported the existence of an androgenic gland distinct from the testes in decapod crabs. She a,lso has reported that this gland is responsible for the development and maintenance of both the primary and secondary male sex characteristics in crustaceans (CharniauxCotton, 1960). The androgenic gland has been described in various crustaceans (Charniaux-Cotton, 1960) as a small gland generally associated with the subterminal end of the vas deferens. Charniaux-Cotton (19%) specifically noted the location of the gland in the lobster, Homarus vulgaris, an animal very similar to the American lobster, H. umericanus. No simple method for detecting the gland has been reported. * Steroid nomenclature : androstenedione = androst-4-ene-3,17-dione : testosterone = 17&hydrosyandrost-4-ene-3-one ; ll-hydroxytestosterone = 11~,17,Gdihydroxyandrost-4-ene-3-one; ll-ketoteetoeterone = 17,E-hydrosyandrost-4-ene-3,l~-dion~: adrcnostcrone = androst-4-cne-.3,11,17-trione; rpitestosterone = 17a-hydroxyandroet-4-em-3-one.

The current general opinion is that. crustacean androgenic gland produces a proteinaceous hormone (Charniaux-Cotton, 1962; King, 1964). However, preliminary work reported by Sarojini (1964) indicates that a sterol fraction, or at least the nonsaponifiable lipid fraction, extractable from the androgenic gland of Cic;ypoda platytnris can mimic the effect of androgenie gland transplants on female crabs. In addition, Sarojini (1963) presented cvidence that injected testosterone could produce the primary but not the seconda,ry male sex characteristics in the crab. Other evidence implicates steroids AS normal crustacean constituents. ‘Estrogen (Donahue, 1948, 1952, and 1957) and specifically 17B-estradiol (T&k, 1961) has been detected in lobster eggs, :and oketolic steroids may be present in C~IIRtacean ovaries iI3rodzicki, 1963). Finally, ecdysonc-like compounds have been isolated from crustacean sources (Gailego and Mcn6nclez, 1959; Horn. Et nl., 1966; Hampshire and Korn7 1966; nrb%n wntl Skinner. 1960).

319

320

GILGAN

AND

The ability to oxidize testosterone to androstenedione has been detected in sperm of the oyster, sand dollar, and sea urchin (Hathaway, 1965). Histochemical evidence suggests that both 17P-hydroxysteroid and A5-3fl-hydroxysteroid dehydrogenases may occur in certain marine invertebrate tissues (Mori, et al., 1964; 1965). Despite the known presence of substances with steroid ring systems in crustaceans, evidence of the ability of crustacean tissues to transform known steroids to welldefined products is lacking. This paper represents a preliminary attempt to overcome this deficiency. The principle purpose of the work reported here is to see if tissues of the American lobster, and in particular androgenie gland or testis, can transform any of the known vertebrate steroid hormones. In view of the work of Sarojini (1963) the transformation of androstenedione to testosterone was selected for study. When preliminary work indicated that several lobster tissues were able to effect the transformation, it was considered desirable to compare the activity of various tissues. MATERIALS

AND

METHODS

ANIMALS

All lobsters were intermolt and had been held in captivity for at least one month. The tissue samples for the first comparison series were from animals obtained from Cape Breton, N.S., and held in flowing sea water at 3~3°C (normal deepwater temperature) until late August. The tissues for the second series were from Pugwash, N.S., animals held in 13”C, carbon filtered, recirculated sea water until eariy Oetober. TISSUE

IDLER

the normally invisible androgenic gland, was used for incubation. Testis samples were taken from animals with well-developed vas deferens. All weighed tissue samples were preincubated at room temperature, ca. 25”C, for 1.5 hours in lobster saline. Three of the four vas deferens samples were preincubated in 2 ml of dye solution. This solution was prepared by dissolving 0.1 ml of a propylene glycol solution of l-2 mg dinitro blue tetrazolium (Dinitro BT, Dajac Laboratories, Philadelphia 24, Penn.) in 7 ml of lobster saline. After about 15 minutes a tissue apparently identical to reported androgenic gland became visible as a surface layer of purple chains of cells extending between (ea.) 0.5 and 2 cm of the basal end of the vas deferens. The location of the gland was variable and the tissue was not visible without dye treatment. After about 15 minutes the tissue was transferred to fresh lobster saline at room temperature (25-28°C) and the androgenic gland was removed from two tissue samples by microdissection. In addition, one of the glandless samples was split and the mucosa removed with a steel spatula. The treated sa,mples were then reweighed. INCUBATION

METHOD

All tissues were cut into small pieces with scissors and added to a mixture consisting of about 0.1 PC C4-“Cl androstenedione (200,000 dpm, 1 mc/6.34 mgl in 0.1 ml propylene glycol, 2-ml homarus saline contxining sodium glucose-6phosphate (12 mg) NAO (nicotinamide adenine dinucleotide, 3 mg) and nicotinamide (2 mg). The tissue fragments and solution were then stirred slowly with a magnetic bar for 3 hours at room temperature. At the end of the reaction about 0.05 PC 11,2-3H1 testosterone (86,900 dpm) in 0.05 ml benzene-methanol (9: 1 v/v), 10 pg carrier testosterone and 2 ml methanol were added. A tissue-less 14-“Cl androstenedione control was treated identically to the experimental samples in the incubat.ion and subsequent extractions.

PREPARATION

All animals were anaesthetized prior to tissue removal with 8-ml isobutanol per liter of sea water (Foley et al., 1966). The hepatopancreas, testis, vas deferens, and anterior tail muscle were quickly excised and chilled to ice temperature in lobster saline (Presser, 1950) ,modified m that 20 ml 0.05 M tris hydroxymethyl aminomethane, pH 7.4, per liter of saline, was substituted for borate buffer. Tissue samples of approximately equal wet weight (150 mg) were prepared. Only the muscular part of the vas deferens, known to carry

EXTRACTION

In each case the complete suspension was extracted in a test tube with 3 x 4-ml dichloromethane. Mixing for each extraction m-as achieved by 2 x 30-second agitation with a vortex mixer. Emulsions were broken by centrifugation. The combined dichloromethane extracts were washed with 1 x 1.2 ml 0.5F NaCO,, 1 x 1.2 ml water, 1 x 1.2 ml 1 N acetic acid and, finally, 3 x 1% ml water. The solutions were evaporated to dryness with a stream of nitrogen and the residues stored in a vacuum desiccator overnight.

CONVERSION

THIN-LAYER

OF

ANDROSTENEDIOWE

CHROMATOGRAPHY

TO

(TLC)

kJTHENTICATIOS

OF TESTOSTERONE

SAMPLES

Vas deferens -f androgenic Hepatopancreas

‘Pestis

?S

1 BY LOBSTER

TISKES

Total “C-testosterone

Per cent conveision of androstenedione to testosterone

13s,ooo 139,000 85,400

68.6 69.5 42.6 16.6 13.5 9.5 68.3 75.5

(dpm)

33,100

Tail muscle

TESTIS MKABOUW

32B

There can be little doubt that the product isolated from the testis incubation mixture was testosterone. The compoucd could be recrystallized after TLC purXcation with authentic 3H-labeled and cold testosterone with little variation in the 92 to “H ratio. The material had the

SYNTHESIS synthesizedn

gland

THE

TISSUE

RESULTS

TABLE

l’issue

OF

LOBSTER

A sample of the TLC purified testosterosle VFHS concentrated and applied to Whatman No. 1 paper. Authentic testosterone and trstostcrone samples were spotted iLt pius epitestosterone separate origins. -4fter 3 hours equilibration tht: chromatogram was developed for 18.5 hours >jt 25°C in rL-heptane saturated with methanol:aater (4:l v/v). The system readily scparatrd t&o;terone from epitestosterone. The testosterone zone was eluted and a sample counted for ‘H-l% radioactivity. The solution wa.s evaporated with nitrogen and dissolved ir, 0.3-ml glacial acetic acid containing 80’ pg CrG,. A& 40 minutes the solution was evaporat-tl and the residue dissolved in methanol. The sample was spotted on Whatman No. 1 and, aitcr equilibration as before, developed for 6 hours in ihc above mentioned system. Authentic androstenedione was readily separated from testosterone. The, “C-zone which corresponded exactly wi: h (2arriprauthentic and oxidized testostcronc androstenedione samples, was &ted and counted again as above. The eluted C”H-“Cl androstenedionc sitmpie was, added to 3 ,mg authentic androetenedione and crystallized three times from acetone. Samples were counted after each crystallization for ratlo determinations, as before, to achieve a msxirn:rm counting error of 2.7% at 95% confidcncc.

An aliquot cf each of the “testosterone” samples was added to about 10 mg of authentic testosterone and the material recrystallized twice from methanol-water and once from toluenehesane. A4fter each crystallization the sample was dissolved in and diluted to a specific volume with methanol. Samples were then removed for “H-W counting as above. The resultant ratios appear in Table 2.

TESTOSTERONE

BY

TESTOSTERONE

Silica gel GF,, (Merck) 20 x 20-cm plates were poured 0.5 mm thick and dried one-half hour at llO-120°C. A cooled plate was then spotted, generally in two spots, with the whole of each sample. The hepatopancreases, and to some extent testis, extracts were very oily and therefore were spotted at several origins. The plates were then twice developed 15 cm above the origin with n-hexane:ethyl acetate (80:20 ml) as described by Hdler and Truscott (1966) and once wit.h diehloromethanc : acetone (80: 20 ml). This last solvent system readily separated Il-hydroxytestosterone, il-ketotcstosterone, adrenosterone. lip-hydroxy-androstenedione, androstenedione and testosterone. The separation of epitestosterone and testosterone was incomplete. Upon drying, the zones visible in short wavelength (ca. 250 rnp) ultraviolet light were removed and eluted with methanol: dichloromethane 1:9. The solvent was then evaporated and the residue dissolved in 1.25,ml methanol. Samples (25 ~1) were counted for double-label in glass s&tillation vials of Liquifluor scintillation misture to an error of less than 27~ at 95% confidence. From the recovered ‘H-testosterone the per cent recovery was calculated and hence the amount of testosterone produced in the incubation mixture (see Table 1). To check reproducibility thp experiment was repeated. PLI~CRY~T.~LLIZATION

TESTOSTEROXE

27,200 19,000 137,000 151,000

a Based on recoveries of !1,23Hj testosterone following

TLC.

Dpm test&mom synthetizadjn~ tissue

322

GILGAN

AND

TABLE 2 OF 3H, W-TESTOSTERONE ISOLATED BY TLC

RECRYSTALLIZATION

Recrystallization 1=t

Tissue

Vas deferens (V.D.) V.D. + nitro BT V.D. + nitro BT - androgenie gland (A.G.) V.D. + nitro BT - A.G. - mucosa Hepatopancreas Tail muscle Testis

2nd

3rd

3.10 3.01 2.08

3.07 3.06 2.03

2.98 2.97 2.12

0.608

0.570

0.571

2.15 0.565 3.67

2.11 0.551 3.58

2.11 0.526 3.55

a For any tissue the maximum mean value was 4.370.

deviation

from

the

mobility of testosterone in a paper chromatographic system known to separate testosterone from epitestosterone, the expected contaminant if the enzymatic reduction was nonspecific. It was then oxidized to a material which chromatographed as androstenedione in the same paper chromatographic high resolution system. The l”C to 3H count ratios after chromatography as testosterone and as androstenedione were 3.39 and 3.66, respectively. The fact that this ratio remained approximately the same after repeated chromatography and also that the strip radioactivity scans showed single peaks in each chromatogram, which corresponded exactly in each case to the position of the cold carrier and to the expected compound, would indicate that the original compound was radio-chemically pure and was, in fact, testosterone. When the chromatographed oxidation product was recrystallized three times with cold androstenedione the 14(S3H radioactivity ratios were 3.24, 3.48, and 3.58. The maximum per cent deviation from the average of the three values was 5.5. The mother liquors and the final crystals were recombined and recrystallized for a second time to give ratios of 3.58, 3.46, and 3.48 with maximum deviation from the mean of 2.370. The nature of the material isolated from

IDLER

the incubation mixtures other than those of the testis was less rigorously established as testosterone but, since the recrystallization ratios remained essentially constant (Table %), little doubt remains that at least the bulk of each TLC purified fraction was testosterone. That is, the TLC purification was highly successful in the case of the testosterone synthesized by the testis, as shown by subsequent chromatography and recrystallization, and would therefore most likely be as successful with the other samples. An examination of the data in Table 1 shows that the highest per cent conversion of androstenedione to testosterone was achieved by testis while that of the intact vas deferens (including attached androgenie gland) was nearly as high. As might be expected muscle tissues showed considerably lower honversion. The hepatopancreas and tail muscle tissue samples were included in the comparison because preliminary investigation suggested that reductase activity was present in all lobster tissues. It can be seen in Table 1 that botk tissues apparently have some activity. However if conditions were selected, the synthetic ability of these two tissues would likely appear Iess favorable in comparison to that of the sexual tissues. The small activities observed in these tissues were not artifacts of the androstenedione sample, as the tissueless control contributed no significant W-radioactivity to the testosterone fraction. A closer examination of the per cent conversions achieved by the vas deferens with and without dye, after removal of specific tissues, is presented in Table 3. It can be seen that the dye had little effect on the total testosterone produced. On the other hand, removal of the androgenic gland or mucosal tissue caused a drastic decrease in the amount of substrate reduced. Of these two, the androgenic gland caused a disproportionately high conversion considering its small mass. DISCUSSION

Little is definitely known as to how best to manipulate and to incubate crustacean

CXXWERSION

OF

ANDROSTENEDIONE

TO

TABLE I%-TESTOSTEROICE

FORMED

BY THE BEFORE Total terone

Tissue

sample

Vas deferens (V.D.) gland (A.G.)

with

V.D.

with

A.G.

+ dye without

A.G.

V.D.

+ dye without,

A.G.

a Based on recoveries b Wet weight.

and mucosa

of [1,23H]

VAS

AFTER

DYE

YZ-a testossynthesized (&ml

testosterone

138,000 129,000 138,000 83,100 93,400 21,300 26,300 following

tissue. Thus the methods used were primarily adopted because they proved successful with other marine forms. It has not yet been possible to isolate lobster androgenic gland without detecting it with some type of stain, thus it was impossible to directry determine that the dye and preincubation were not adversely affecting the tissue. Hence the approach used here was that of the effect of tissue removal on total tissue activity. In these preliminary experiments the large variation in the per cent conversion of androstenedione to testosterone in the various preparations does not permit meaningful comparison of the specific activities of the tissues. It must be remembered that, the actual amount of substrate in each ease is exeremely small and therefore the enzyme activity will be limited in the tissues not by the amount of enzyme present but by the availability of substrate. Before direct comparison of the enzyme levels in the various tissues can be accurately assessed the kinetics of the system must be studied. The enzyme activity of hepatopancreas and muscle tissue might not appear to be as high in comparison to the sexual tissues if optimum conditions were achieved. It would seem that lobster tissues are capable of at least one of the steroid conversions of the vertebrates. The significance of the ability to reduce androstenedione specifically to testosterone, most

BY

LOBSTER

TISSIJE

ASSOCIATES

Trssc~s

323

3

LOBSTER

139,000

androgenic

+ dye

V.D.

AND

TESTOSTEROXE

DEFERENS

AND

TREATWENT

Weight6 of tissue removed (mg)

Decrease radioactivity

in total (dpm)

,% of. orif;g;;zsue

yJ;;y actxey

1

-

10,000 0

13 12 47 69

56,000 45,000 118,000 112,000

-

_-

0 0 8 a 26 41

7 0 40 33 91 81

TLC.

notably by the androgenic gland and testis, is not known. As was mentioned before, it is generally presumed that the male hormone is proteinaceous (Charniaux-Cotton, 1962 ; King, 1964). The work of Sarojini (1963 ; 1964) mentioned earlier indicates, however, that at least part of the male hormone activity may be due to a testosterone-like compound. Assuming that. the primary male hormone was ~roteina~e~~s~ this need not exclude steroid compounds from playing a secondary role, such as is suggested by the work of Donahue (1965) in that injected estradiol benzoate inhibited molting. The above evidence shows that erustaceans can transform androstenedione to testosterone but thus far, efforts to detect testosterone in lobster blood have been unsuccessful (unpublished experiments j . The data presented herein indicate that a more intensive investigation of the probIem is warra’nted. REFERENCES 8. (1963). Localization of lipids and a-ketolie steroids in the ovary of Crus~ac~a. Folia Histochem. Cytochem. l(2), 259468. CHARNIAUX-COTTON. H. (1956). Existence d’uc organe analogue ?I la “glande androgilne” chcz un Pagure et un Crahe. Corn@. Rend. 143, 1168-69. CHPLRNIAUX-COTTON, R. (1958). La glRn$e andrug&e de quelques crusttacks decapodes :tt particuli6rement de Lysmata seticauduata, es&e BRODZICKI,

324

GILGAN

& hermaphrodisme protkrandrique fonctionnel. Compt. Rend. 246, 2814G?817. CHARNIAUX-COTTON, H. (1960). Sex determination. In “The Physiology of Crustacea” (T. H. Waterman, ed.), Vol. 1, pp. 411447. Acade,mic Press, New York. CHARNIAUX-COTTON, H. (1962). Androgenic gland of crustaceans. Gen. Comp. Endocrinol. Suppl. 1, 241-247. DONAHUE, J. K. (1948). Fluorimetric and biological determination of estrogens in the eggs of the american lobster (Homarus americanus). Proc. Sot. Exptl. Biol. Med. 69, 179L181. DONAHUE, J. K. (1952). Studies on ecdysis in the american lobster (Homarus americanus). I. The ,lobster egg as a source of estrogenic hormone. State of Maine Dept. of Sea and Shore Fisheries, Res. Bull. No. 8. DONAHUE, J. K. (1955). Studies on ecdysis in the american lobster (Homarus americanus). IV. Estrogenic hormone as a possible moultinhibitor in the egg-bearing female. State of Maine Dept. of Sea and Shore Fisheries, Res. Bull. No. 24. DONAHUE, J. K. (1957). Chromatographic identification of lobster egg estrogen. State of Maine Dept. of Sea and Shore Fisheries, Res. Bull. No. 28. FOLEY, D. M., STEWART, J. E., AND HOLLEY, R. A. (1966). Isobutyl alcohol and methyl pentynol as general anaesthetics for the lobster, Homarus americanus Mime Edwards. Can. J. Zool. 44, 141-143. GALLEGO, P. M., AND MEN~NDEZ, D. S. (1959). Hormonas en crustaceos. Rev. Espan. Fisiol. 15, 263-268. HAMPSHIRE, F., AND HORN, D. H. S. (1966). Structure of crustecdysone, a crustacean moulting hormone. Chem. Commun. 1966(Z), 37-38.

AND

IDLER

HATHAWAY, R. R. (1965). Conversion of estradiol17p by sperm preparations of sea urchins and oysters. Gen. Comp. Endocrinol. 5, 504-508. HORN, D. H. S., MIDDLETON, E. J., WUNDERLICH, J. A., AND HAMPSHIRE, F. (1966). Identity of the moulting hormones of insects and crustaceans. Chem. Commun. 1966(U), 339-40. IDLER, D. R., AND TRUSCOTT, B. (1966). Identification and quantification of testosterone in peripheral plasma of skate. Gen. Comp. Endocrinoz. 7, 375383. KARLSON, P., AND SKINNER, D. M. (1960). Attempted extra&ion of crustacean moulting hormones from isolated Y-organs. Nature 185, 543-544. KING, D. S. (1964). Fine structure of the androgenie gland of the crab, Pnchygrapsus crassipes. Gen. Comp. Endocrinol. 4, 533-544. LISK, R. D. (1961). Estradiol-17,8 in the eggs of the american lobster, Homarus americanus. Can. J. Biochem. and Physiol. 39, 659-663. MORI, K., TAMATE, H., AND IMAI, T. (1964). Presence of A’-3,&hydroxysteroid dehydrogenase activity in the tissues of maturing oysters. T6hoku J. Agri. Res. 15, 269-277. MORI, K., TAN~TE, H., AND 1x.41: T. (1965). Presence of 17,8-hydroxysteroid drhydrogenase activity in the tissues of maturing oysters. TGhoku J. Agri. Res. 16, 147-157. PROSSER, C. L. (1950). In “Comparative Animal Physiology” p. 95. W. B. Saunders Company, Philadelphia. SAROJINI, S. (1963). Comparison of the effects of androgenic hormone and testosterone propionate on the female Ocypod crab. Current Sci. (India) 32(9), 411412. SAROJINI, S. (1964). A note on the chemical nature of crustacean androgenic hormone. Current Sci. (India) 33(Z), 55-56.