Steroids 77 (2012) 1450–1468

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Steroids

journal homepage: www.elsevier .com/locate /s teroids

Review

Do mollusks use vertebrate sex steroids as reproductive hormones? Part I:Critical appraisal of the evidence for the presence, biosynthesis and uptake ofsteroids

Alexander P. ScottCentre for Environment, Fisheries and Aquaculture Science, Barrack Road, Weymouth, Dorset DT4 8UB, UK

a r t i c l e i n f o

Article history:Received 12 June 2012Received in revised form 16 August 2012Accepted 21 August 2012Available online 31 August 2012

Keywords:Vertebrate steroidInvertebrate steroidMolluscBiosynthesisReceptorTissue concentrations

0039-128X Crown Copyright � 2012 Published by Elshttp://dx.doi.org/10.1016/j.steroids.2012.08.009

E-mail address: Alexander.Scott@cefas.co.uk

a b s t r a c t

The consensus view is that vertebrate-type steroids are present in mollusks and perform hormonal roleswhich are similar to those that they play in vertebrates. Although vertebrate steroids can be measured inmolluscan tissues, a key question is ‘Are they formed endogenously or they are picked up from their envi-ronment?’. The present review concludes that there is no convincing evidence for biosynthesis of verte-brate steroids by mollusks. Furthermore, the ‘mollusk’ genome does not contain the genes for keyenzymes that are necessary to transform cholesterol in progressive steps into vertebrate-type steroids;nor does the mollusk genome contain genes for functioning classical nuclear steroid receptors. On theother hand, there is very strong evidence that mollusks are able to absorb vertebrate steroids from theenvironment; and are able to store some of them (by conjugating them to fatty acids) for weeks tomonths. It is notable that the three steroids that have been proposed as functional hormones in mollusks(i.e. progesterone, testosterone and 17b-estradiol) are the same as those of humans. Since humans (andindeed all vertebrates) continuously excrete steroids not just via urine and feces, but via their body sur-face (and, in fish, via the gills), it is impossible to rule out contamination as the sole reason for the pres-ence of vertebrate steroids in mollusks (even in animals kept under supposedly ‘clean laboratoryconditions’). Essentially, the presence of vertebrate steroids in mollusks cannot be taken as reliable evi-dence of either endogenous biosynthesis or of an endocrine role.

Crown Copyright � 2012 Published by Elsevier Inc. Open access under CC BY-NC-ND license.

Contents

1. General introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 14512. Occurrence of steroids in molluscan tissues. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1451

2.1. Seasonal changes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 14512.2. Sex differences. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 14512.3. Evidence that contaminants affect steroid concentrations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 14512.4. Tissue differences . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 14542.5. Studies not included in Table 1 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1454

3. What might explain the presence of vertebrate steroids in mollusks? . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 14544. Could there be problems with the measurement procedures? . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1455

4.1. Lack of specificity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1455

5. Do the animals biosynthesize the steroids themselves? . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1455

5.1. Evidence for a vertebrate-type biosynthetic pathway . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 14555.2. Possible problems with steroid identification . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 14555.3. Incubation procedures . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 14565.4. The problems of low (and unreported) yields . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 14565.5. Do mollusks have cholesterol? . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 14565.6. The first key step in steroid biosynthesis – the cleavage of the side chain of cholesterol . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 14565.7. D5-3b-HSD activity (column 2 in Table 2). . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 14585.8. C17,20-lyase (column 3 in Table 2) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 14585.9. 17b-hydroxysteroid dehydrogenase (17b-HSD) activity (column 4 in Table 2) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1459

evier Inc. Open access under CC BY-NC-ND license.

A.P. Scott / Steroids 77 (2012) 1450–1468 1451

5.10. 5a-reductase (column 6 in Table 2). . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 14595.11. CYP19 (aromatase; column 5 in Table 2). . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 14595.12. A strong streak of anthropomorphism in mollusk studies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 14595.13. Interim conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1459

6. The animals pick up the steroids from the environment?. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1460

6.1. Possible sources of steroids in the environment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 14606.2. Experimental evidence that mollusks can take up and store exogenous vertebrate steroids . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 14606.3. What is the point of esterification? . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 14616.4. What can explain the presence of steroids in laboratory-reared animals? . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 14616.5. Looking for steroids that are unlikely to have been formed endogenously. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1461

7. The evidence for steroid receptors in mollusks . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1462

7.1. Presence of classical nuclear receptors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 14627.2. Another class of nuclear receptor?. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 14627.3. Could it be a ‘non-classical’ membrane-bound receptor?. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 14647.4. Could there be binding proteins that are not receptors? . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 14647.5. Does the presence of a receptor necessarily mean the ligand has to be endogenous?. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 14647.6. Is a receptor necessary to elicit effects? . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1464

8. Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 14649. Future directions? . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1465

Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1465References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1465

1. General introduction

The question as to whether vertebrate steroids act as hormonesin mollusks is a very important one. Many compounds that behavelike vertebrate estrogens are present in the environment (so-called‘endocrine disrupters’), and if they act in the same way in mollusksas they do in vertebrates [1] then there is genuine cause for con-cern. The first reports of the existence of vertebrate-type steroidsin mollusks appeared in the 1950s [2,3]. Since then, all but a hand-ful of the 200+ scientific papers and reviews that have been pub-lished in this area have a positive message (i.e. they concludeeither that mollusks contain vertebrate steroids, are able to biosyn-thesize them de novo, appear to contain steroid receptor-like bind-ing activity or respond in one way or another when exposed tovertebrate steroids). The sheer ‘weight of evidence’ would seem,on the surface, to make it an ‘open and shut’ case that vertebratesteroids are an important component of molluscan endocrinology.However, if one looks beyond the headline claims (i.e. essentiallywhat is written in the titles and abstracts of many of the papers),a different story emerges – one in which most, if not all, of the po-sitive evidence can be seen to be rather weak (in that the data areopen to alternative interpretations). This review paper deals withthe strength of the evidence for the presence of steroids in mol-lusks, for their biosynthesis and for the presence of steroid recep-tors. Another review paper [4] deals with the strength of evidencefor the biological actions of vertebrate steroids on mollusks. Mostof the literature that has been reviewed was obtained from the ref-erence lists of previous reviews and key papers in the field. Morerecent papers were picked up by searching Scopus and Web of Sci-ence for papers that cited certain key papers (plus keywords suchas ‘mollusk’ and ‘steroid’). A final search was made in March, 2012.

2. Occurrence of steroids in molluscan tissues

There are numerous (>50) publications that report the presenceof typical vertebrate steroids such as testosterone (T), 17b-estra-diol (E2) and progesterone (P) in a wide range of mollusks (Table1). In Table 1, concentrations are all shown as an approximaterange (or in some cases a single value); are quoted as pg g�1 wetweight of tissue; and are shown for free as well as esterified (seeSection 6) steroids, when both have been measured. Mode of assayis briefly indicated. The final three columns indicate whether thesteroid concentrations were found to differ by season, sex or pollu-

tion. An indication is also given of the magnitude of these changes– as the fact that steroid concentrations change (with season inparticular) is frequently cited as evidence that the steroids perhapsfunction as reproductive hormones as they do in vertebrates.

2.1. Seasonal changes

One argument for the involvement of vertebrate steroids inmollusk reproduction is that, in many studies (column 7 in Table 1),the concentration of steroids (and this mostly refers to free ste-roids) varies significantly with the time of year. Furthermore, thesechanges have been claimed to match reproductive events. How-ever, neither the size of the change nor any apparent associationof a peak with reproductive events can be considered as incontro-vertible evidence that the animals use those steroids as hormones.Equally plausible alternative hypotheses are, firstly, that the con-centrations of the steroids just reflect the levels of steroids thatare available to be absorbed from the water (see Section 6) or sec-ondly, any association is purely by chance.

2.2. Sex differences

Very little difference has been found between steroid concen-trations in males and females (column 8 in Table 1). However, thisneither proves nor disproves that T and E2 are hormones in mol-lusks. In most teleost fishes, both steroids are found in the bloodplasma of females [5]; and in a few species, both steroids also occurin male plasma [6].

2.3. Evidence that contaminants affect steroid concentrations

A few experiments have been carried out to determine whethercontaminants, especially tributyl tin (TBT), affect steroid concen-trations (column 9 in Table 1). The effects have in most cases beenminimal. Other studies have involved sampling (or deliberatelyplacing) animals at contaminated or pristine sites. Although thesehave tended to yield larger differences in steroid concentrationsthan purely laboratory experiments, it is impossible to rule outthat the differences were due to the amounts of steroids that theanimals might have taken up from the environment (see Section 6).

Table 1Steroid concentrations that have been reported in molluscan tissues.

Species Source Tissue Steroid Assay Levels (pg g�1) Seasonality/stage differences? Sex differences? Contamination effects?

Giant ramshorn snail,Marisa cornuarietis[75]

Lab.Stock

Whole tissue T (ester) RIA 800 (2000–30,000) 4-fold Yes for esterified T and E2(males 3–5 times higher)

TBT for 100 days reduced esterified T and E2in females onlyE2 (ester) 100 (6000–90,000)

Cuttlefish, Sepia officinalis[40]

Wild Hemolymph T RIA 150 – No –Estrogen 10 ?

Tissue T 10,000–20,000 –

Dogwhelk Nucella lapillus[81]

Wild/tank

Whole tissue(female only)

T (ester) RIA 1000 (10,000) – – Slight elevation of free by TBTE2 1000 2-fold elevation by TBT + CPA

Dogwhelk Nucella lapillus[117]

Wild/tank

Whole tissue offemales

T RIA 1000–7000 – – Slight effect of TBTE2 20–500P 1000

Dogwhelks Nucellalapillus and Hiniareticulata [118]

Wild/tank

Whole tissue offemales only

T RIA 600–1200 – – 2-fold difference between imposex stages 0and 4

Eastern mud snail,Ilyanassa obsoleta[119]

Wild Gonad/viscera T (ester) RIA 25,000 (100,000) 4-fold (fem) to 12-fold (male),but same pattern in both sexes

10-fold higher in femalesat dormant phase

–

E2 (ester) 500 (3000) 10-fold No

Eastern mud snail,Ilyanassa obsoleta[68,69]

Wild Whole body T RIA 2000–45,000 (highest levels in April andNovember coincide with lowesterification)

10-fold No –

Eastern mud snail,Ilyanassa obsoleta[68,78]

Wild/tank

Whole body T (ester) RIA 2500 (25,000) – – Slight increase in free T after 3 months ofTBT

Garden snail, Helixaspersa [82]

Culture Gonad T RIA andGC–MS

8000–28,000 3-fold higher in juveniles – –Ad 2500 3-fold higher in juvenilesP 3000 2-fold higher in adults

Hemolymph T 300 NoAd 20 NoP 1000–6000 5-fold higher in adults

Giant African land snail,Achatina fulica [17]

Lab Hemolymph T RIA 30–700 – Yes –E2 0–2000 YesP 440–8000 YesAd 0–100 YesCortisol 300–2000 No

Grooved carpet shell,Ruditapes decussatus[120]

Culture Whole tissue T RIA 120,000 – – After 5 weeks in TBT-contaminated harbor, Trose and E2 fellE2 5000–20,000

Grooved carpet shell,Ruditapes decussatus[121]

Wild/tank

Whole tissueminus digestivegland

T RIA 100–800 – – Increased by TBT exposureE2 <0.2

Grooved carpet shell,Ruditapes decussatus[122]

Wild Gonad T RIA 40–400 8-fold Slight –E2 10–240 20-foldP 200–2500 15-fold

Japanese scallop,Paticopecten yessoensis[56]

Wild Gonad E2 EC 1500–4500 3-fold No –E1 <500

Manilla carpet shell,Tapes philippinarum[123]

Wild Whole body T RIA 100–200 2-fold Slight –E2 100–300 3-foldP 600–1600 3-fold

1452A

.P.Scott/Steroids77

(2012)1450–

1468

Table 1 (continued)

Species Source Tissue Steroid Assay Levels (pg g�1) Seasonality/stage differences? Sex differences? Contamination effects?

Mussel, Mytilus edulis[42]

Wild Mantle + gonad T RIA 1400–40,000 40-fold in males 10-fold in males at latestage

–

E2 4500 Only present in late stage NoE1 35,000 Only present in late stages No

Mussel, Mytilus edulis[124]

Wild Whole body P GC–MS 5,000–40,000 10-fold No –Gonad RIA 1,500–4,000 3-fold

Mussel, Mytilus edulis[125]

Wild Whole body T GC–MS 200–600 3-fold No -E2 20–40 2-foldP 500–5000 10-foldE1 60 No changeAd 1000–3500 3.5-fold

Mussel, Mytilus edulis [8] Wild Gonad T (ester) RIA 3500 (4000–12000) – – 3-fold increase in esterified steroids ingonads after exposure to North Sea Oil for3 weeks

E2 (ester) 1000 (4000–11000)Peripheral tissue T (ester) 150 (2000–5000)

E2 (ester) 20 (2000)

Mussel, Mytilusgalloprovincalis [73]

Wild Whole tissue E2 (ester) RIA 800 (10,000) – – Not affected by addition of T to water for5 days

Mussel, Mytilusgalloprovincalis [126]

Wild Whole tissue T (ester) RIA 1000 (1500) – – Not affected by addition of E2 to water for5 days

Mussel, Mytilusgalloprovincalis [127]

Wild Mantle + gonad E2 RIA <10–100 (but from pooled samples only) 10-fold – –

Mussel, Mytilus spp. [25] Wild Digestive gland P GC–MS <400–7000 >15-fold No –Preg 500–9000 18-fold NoT, E1, E2,Ad, DHT,DHA

All <400 – No

Octopus, Octopus vulgaris[9]

Wild Reproductivetissue of malesonly

T RIA/EIAandHPLC

2500–5000 – – –200–10001000–5000

Octopus, Octopus vulgaris[128]

Wild/Tank

Ovary E2 RIA 25–200 4-fold elevation of bothsteroids in May only (i.e. nocycle)

– –

Pacific oyster, Crassostreagigas [50]

Culture Gonad E2 RIA 0–1500 >20-fold 3-fold in females –E1 0–300 >20-fold

Peppery furrow shell,Scrobicularia plana[129]

Wild Gonad T EIA 60–240 4-fold Yes Yes T higher in males at two sites and Phigher in females at other siteE2 100–300 3-fold No

P 200–2800 5-fold Yes

Purple dye murex, Bolinusbrandaris [130]

Wild Digestivegland + gonad

T RIA 800 – No E2 levels much lower at TBT-contaminatedsiteE2 <5–250

Rockshell, Thais clavigera[7,24]

Wild Testis and ovary T EIA &GC–MS

1000 – No No

Scallop, Patinopectenyessoensis [50]

Culture Gonad E2 EC 400–1100 No 2-fold higher in femalesonly

–Wild Gonad E1 200 No

Soft-shell clam, Myaarenaria [131]

Wild Gonad T EIA & 40 Slight (2-fold) Slightly higher in females –E2 GC–MS 300

Soft-shell clam, Myaarenaria [132]

Wild Gonad P EIA &GC–MS

4000 Slight (1.5-fold) No –

(continued on next page)

A.P.Scott/Steroids

77(2012)

1450–1468

1453

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1454 A.P. Scott / Steroids 77 (2012) 1450–1468

2.4. Tissue differences

Very few studies seem to have examined tissue distribution ofsteroids. In the rockshell, Thais clavigera [7], the gonads had fivetimes higher concentrations of T and E2 than in the remaining tis-sues. In the mussel, Mytilus edulis [8], the concentrations of free(but not of esterified) T and E2 were 18- to 91-fold, respectively,higher in the gonads than in the remaining tissue. In the octopus,Octopus vulgaris [9], steroid levels were also more abundant inreproductive tissue (i.e. testis vas deferens, seminal vesicle andprostate) than in non-reproductive tissues. Steroid concentrationsin hemolymph (the mollusk equivalent of ‘plasma’) have in generalbeen found to be much lower than those that can be extracted fromtissues (see Table 1). It would be tempting to conclude that if ste-roid concentrations in gonads are higher than those in other tissuesit means either that they are synthesized by the gonads [8] or thatthey must have a role in reproduction. However, a plausible alter-native explanation is that steroids are preferentially accumulatedby the gonads because of their generally higher content of fatand protein (to which steroids could be non-specifically absorbed).

2.5. Studies not included in Table 1

There are at least seven studies on the New Zealand mudsnail,Potamopyrgus antipodarum that have not been included in Table 1,because steroid concentrations in these studies were only quotedas fmol individual�1 or pg individual�1 (and not pg g�1 tissue asin Table 1) and, also, the authors in most cases only measured total(i.e. free + ester) steroid concentrations [10–16].

There are, surprisingly, only two studies involving in vitro re-lease of steroids by gonad tissues from mollusks. These are not in-cluded in Table 1 either. In the giant African land snail, Achatinafulica [17], steroids were found to be released by ovotestis tissue.More P and T release was found by male phase gonads than by fe-male phase gonads and vice versa for E2. No cortisol release wasfound in vitro (despite evidence for its presence in hemolymph).In O. vulgaris [18], ovarian follicles and spermatozoa were bothshown to release immunoactive T (10-fold), E2 (2-fold) andP (4-fold) in vitro, over a period of 90 min, in response to stimula-tion with a peptide similar to vertebrate gonadotropin-releasinghormone.

Finally there are several early studies in which steroids wereputatively identified by bioassay or chromatography coupled withmicrochemistry. For example, in the periwinkle, Littorina littorea,compounds that had estrogenic and androgenic effects in mam-mals were extracted from the gonads [3]. In the slug, Arion ater ru-fus [19], the presence of ‘estrogens’ was revealed by TLC andmicrochemistry in spermatheca that had been incubated in vitro.There were no signs of precursors such as T and androstenedione(Ad), however and authors suggested that these steroids may havealready been present in the tissues (i.e. not synthesized de novo).Another study identified 11-ketotestosterone (KT; an androgen inteleosts) by the same methods in the same species [20]. However,later studies showed no evidence for biosynthesis of this steroid inthe banana slug, Ariolimax californicus [21]. In the scallop, Pectenmaximus [22], P was tentatively identified by its position on TLC.None of these procedures are specific enough for any of the identi-fications to be accepted as ‘definitive’.

3. What might explain the presence of vertebrate steroids inmollusks?

As we have seen, there are a surprisingly large number of stud-ies that report the presence of vertebrate-type (predominantly hu-man) steroids in the tissues of mollusks. To explain the presence of

A.P. Scott / Steroids 77 (2012) 1450–1468 1455

these steroids in mollusk tissues, there are essentially threehypotheses – none of them mutually exclusive:

A. Problems exist with the measurement procedures.B. The animals biosynthesize the steroids themselves.C. The animals sequester the steroids from the environment

4. Could there be problems with the measurement procedures?

4.1. Lack of specificity

The main methodology that has been used to measure steroidsis immunoassay. Immunoassays are notoriously liable to interfer-ence termed ‘matrix effects’ in which compounds that have beenextracted along with the steroids affect the affinity of the antibodyand thus alter the overall binding to the labeled ligand (and hencethe apparent concentration of steroid). Also, no steroid antibodiesare 100% specific and some compounds (usually closely related ste-roids; and conjugates of the steroid) are able to displace the labeledsteroid to the same or a lesser extent. Very few of the published pa-pers in the mollusk field include any attempt to even partiallycharacterize the purported steroids that were being measured.One paper that did [23] found that most of the E2 measured byradioimmunoassay (RIA) in tissue extracts of M. edulis, ran in thevoid volume of a high performance liquid chromatography (HPLC)column and that only 10% actually ran in the expected elution po-sition of E2. The latter peak, however, was definitively identified asE2 by mass spectrometry. Another study [24] showed that, whenenzyme immunoassay (EIA) was used to measure T in crude tissueextracts, it gave at least 10-fold higher readings than gas chroma-tography coupled with mass spectrometry (GC–MS). However aftercarrying out several purification steps, the EIA gave the same read-ing as GC–MS.

In addition to the possibility of matrix effects and cross-reac-tion, there are plenty of opportunities for human error in assaymeasurements. However, without access to data sheets or labora-tory workbooks, it is impossible to establish that such errors mightor might not have taken place. There are enough studies on steroidmeasurement in mollusks, however, that have been repeated andindependently verified and have used highly specific detectionmethods (e.g. GC–MS), that one must accept that vertebrate ste-roids can definitely (though, it must be stressed, not always [25]be detected in molluscan tissues. Also, it seems very clear that sub-stantial amounts of T and E2 in particular are present in the form offatty acid esters (in those species that have been investigated).

5. Do the animals biosynthesize the steroids themselves?

5.1. Evidence for a vertebrate-type biosynthetic pathway

The pathway that leads to the biosynthesis of steroids such as P,T and E2 in humans is well-known and well-characterized (Fig. 1).The conventional way to demonstrate the existence of steroid bio-synthetic pathways is to incubate tissue explants with radioactivesteroid precursors. Although there have not been a huge number ofsuch studies in mollusks (Table 2), they are often cited in supportof the claim that mollusks are able to make their own vertebrate-type steroids. So how robust is the evidence?

There is little doubt that mollusk tissues will transform steroidsin vitro. None of the studies in Table 2 have reported zero transfor-mation of added precursors. This is not surprising, as steroids arealso transformed by organisms as diverse as algae, bacteria, otherinvertebrates and higher plants [26–28]. What we need to ask iswhether, in mollusks, any such transformations are:

A. Part of a specific pathway involved in the formation of thesame steroids that are found in vertebrates?

B. Part of a specific pathway involved in the formation of ste-roid hormones that are peculiar to the mollusk in question?

C. Part of a pathway involved in the ‘metabolism’ of steroids?

If the answer is metabolism, then a further question that may beasked is whether the substrate that is transformed by the molluskin question is:

a) an endogenous substrate for which there is a specificenzyme,

b) an exogenous substrate that is transformed by an enzymethat has evolved to deal with a different substratealtogether,

c) an exogenous substrate that is transformed by an enzymethat has evolved to deal with that specific substrate(because, perhaps, that substrate has been encountered inthe environment for millions of years).

One further question [27] (especially when working with filterfeeders) is whether any of the transformations might have beencaused by the algae and bacteria that might have been mixed inwith the tissue.

In biosynthetic studies on mollusks (Table 2), there has been atleast one positive report (defined as a reported yield of >=2% andshown in bold type in the table) that indicates the presence in mol-lusks of all steps of the pathway necessary for the conversion ofpregnenolone (Preg) to P, T and E2. However, these positive obser-vations, especially those on two of the key steps in the cycle (i.e.those catalyzed by D5-3b-hydroxysteroid dehydrogenase [D5-3b-HSD] and C17,20-lyase) have all been on different species. Despitethis, it has been concluded that the whole vertebrate pathwayprobably exists in mollusks [27,29], but is, for some unknown rea-son (that, it is suggested, will be revealed by more research), diffi-cult to demonstrate in an individual species. However, thisconclusion ignores the existence of the far higher proportion ofstudies that have yielded negative, or weakly positive, results(shown in normal type in Table 2) and secondly, it assumes thatthese few positive observations were robust. This is not possibleto establish without independent verification. All sorts of thingscan go wrong with biosynthetic studies that might yield a false po-sitive result (e.g. contamination, misidentification of compounds,mislabeling and miscalculation).

In view of the importance of being able to demonstrate all thesteps of the cycle (using quantitative yield data) within a singlespecies, it is astonishing that, in the last 30 years, only one group[30] has carried out any experiments using either Preg, P or 17-hydroxypregn-4-ene-3,20-dione (17-P) as precursors. That singlestudy showed that the animals were unable to transform Pin vivo. All other biosynthetic studies that have been carried outsince 1981 have been concerned purely with conversions of andro-gens or estrogens (that, as will be made clearer, provide no clues atall as to whether the steroid biosynthesis pathway in mollusks isthe same as that in vertebrates).

5.2. Possible problems with steroid identification

The methodology that investigators have used for identifyingradioactive steroids (column 7 in Table 2) would seem in mostcases to be robust. All have used a combination of chromatography(coupled with microchemistry) and recrystallization to determinethe identity of the steroids (Table 2). However, it should be cau-tioned that such steps are not necessarily foolproof. In a study onthe scallop, Placopecten magellanicus [31], Preg was 34% convertedto a steroid that behaved like androstenedione (Ad) in six different

Fig. 1. Biosynthetic pathway of the main vertebrate steroids that are discussed inthis review – starting from pregnenolone (that is formed directly from cholesterol).Steroids: Preg, pregnenolone; P, progesterone; 17-Preg, 17-hydroxypregnenolone;17-P, 17-hydroxyprogesterone; DHEA, dehydroepiandrosterone; Ad, androstenedi-one; T, Testosterone; E1, estrone; E2, 17b-estradiol. Enzymes: D5-3b-HSD, D5-D4-isomerase) or 3b-hydroxy-D5-steroid:NAD+ 3-oxidoreductase); 17-OHase, 17-hydroxylase; 17b-HSD, 17b-hydroxysteroid dehydrogenase; CYP19, aromatase.Note that 17-hydroxylase and 17,20-lyase activities are both found together onthe one enzyme, CYP17A1.

1456 A.P. Scott / Steroids 77 (2012) 1450–1468

chromatographic systems. However, when it came to the recrystal-lization step, only 0.014% of this activity appeared to behave likeAd. The rest of the activity could not be identified.

Even the technique of ‘recrystallization to constant specificactivity’ is not without its potential problems. If the standard ste-roids are allowed to precipitate (rather than crystallize) this cangive the appearance of homogeneity between labeled compoundand standard. Most steroid chemists will claim indignantly thatthey know what they are doing when carrying out crystallizations(and this is almost certainly true). However, very few people pro-vide evidence that it was actually crystals rather than precipitatesthat they produced during their procedures.

5.3. Incubation procedures

In terms of the methodology used to produce the radioactivesteroids, there are big differences between studies and it is difficultto tell what influence this had on the types or yields of steroids thatwere formed. Steroid precursors have been variously incubatedusing live animals, tissue fragments, minced tissue, thawed tissue,acetone-dried tissue and microsome preparations. Since no proce-dure is accepted as a standard, it is impossible to determine how

choice of procedure might or might not have affected the resultsof any of the studies.

5.4. The problems of low (and unreported) yields

Not all studies have quoted yields (indicated by ‘?’ in Table 2). Insome that have, the conversion rates for critical points in the path-way are so low (<0.01–0.1%) that the question has already beenasked [29,32] as to whether this could possibly result in productionof reported concentrations of vertebrate-type steroids in mollusks.In A. californicus, especially [21], the two key steps (transformationof 3b-D5 ? D4 steroids and of C21 ? C19 steroids), both had <0.04yield (which would in effect mean that 1 mg of Preg would beneeded to produce 1 ng of Ad!

One of the most often-quoted studies is that on the sea hare,Aplysia depilans [33,34]. This study showed that the whole rangeof human steroids (including cortisol) appeared to be formed from14C-labeled acetate, cholesterol, Preg and P by incubation of ace-tone-dried powder of gonads and hepatopancreas. Problems withthis study are that the methods are not described in enough detailto even know whether the authors were using fresh tissue or ace-tone-dried powder for the incubations, the yields were not re-ported and identification was by recrystallization only. Results inthat paper, furthermore, indicate the presence of an impossibleproduct – namely ‘conjugated P’. Conjugation requires the pres-ence of a reactive hydroxyl group (see Section 6) and P has no hy-droxyl groups in its structure.

5.5. Do mollusks have cholesterol?

The raw material from which vertebrate steroids are made ischolesterol. This is found in mollusks in quantities that are morethan sufficient to act as a precursor for steroid synthesis if so re-quired [35]. The only debate is about which species are able to syn-thesize their own cholesterol de novo or have to rely on obtaining itfrom their diet (a question that does not particularly matter to us,as either source could be used for steroid biosynthesis). In theRayed Mediterranean limpet, Patella caerulea, and turban shell,Monodonta turbinata, it was shown [36] that a high proportion ofthe injected 14C-acetate was converted into cholesterol. This wasalso shown in Viviparus fasciatus and L. littorea [37]; A. californicus[38] and A. ater rufus [37]. However, similar experiments with thewhelk, Buccinum undatum [39], and cuttlefish, Sepia officinalis [40]showed zero incorporation. It was suggested that the ability to syn-thesize cholesterol depended on whether the animals were carni-vores or not [39].

It has been speculated that mollusks may use a sterol other thancholesterol as the starting material for steroid synthesis [35].Although several other sterols are known to occur in mollusks[35,41], there is no evidence yet that they are able to be convertedto pregnenolone (i.e. it is still a matter of speculation).

5.6. The first key step in steroid biosynthesis – the cleavage of the sidechain of cholesterol

A key reaction in the synthesis of vertebrate steroids is thecleavage of the side chain of cholesterol (Fig. 2) between C-20and C-22, starting with the addition of an oxygen atom on C20,to form Preg (a 21 carbon compound). The enzyme that performsthis reaction is now commonly referred to as CYP11A1 (with‘CYP’ standing for cytochrome P450). It is difficult, if not impossi-ble, to demonstrate the activity of this enzyme by adding radioac-tive cholesterol to tissue explants in vitro because cholesterol(unlike the other steroids) will not diffuse readily into the cellsand also needs to be actively transported into the mitochondria(where the enzyme is located in vertebrates). This is perhaps the

Table 2The evidence for biosynthesis of vertebrate steroids by mollusks (using 3H- or 14C-labeled steroid precursors).

Species D5-3b-HSD 17-Hydroxylaseplus 17,20-lyase

17b-HSD Aromatase (CYP 19) Other enzymes Methodology

Eastern oyster,Crassostrea virginica[136]

T ? Ad, 14%;E2 ? E1, 93%

Sperm; XT

Atlantic sea scallopPlacopectenmagellanicus [31]

Preg ? 17-P or Ad, noconversion

17-P ? Ad, 0.04%(gonad, but nothepatopancreas)

Ad ? T, noconversion

Gonad orhepatopancreas(minced tissue); PC,TLC, XT

Banana slug, Ariolimaxcalifornicus [21]

Preg ? P, <0.04%;DHEA ? Ad, 0.02%

Preg ? 17-preg,0.01%; 17-P ? DHEA, 0.1%;DHEA ? A, 0.02%

Ad ? T, noconversion; 3a-A(5a) ? 3a,17b-A(5a), 16%

No conversion of Ad to anyestrogens

Ad ? 5a-reducedandrogens, 8%;T ? 5a-reducedandrogens, 60%

Ovotestis(mitochondrial-microsome fraction);PC, TLC, DF, XT

Mussel, Mytilus edulis[42,43]

Preg ? P, 0.4%; 17-Preg ? 17-P, 0.25%

17-P ? A, 0.2% Ad ? T, 2%;E1 ? E2, 50%

Ad ? 5 a -DHT, 0.4% Male and femalegonads (homogenate);TLC, DF, XT

Common periwinkle,Littorina littorea[137]

P ? 17-P, noconversion

P ? 20b-P, 8% Male and femalegonads (homogenate);PC, DF, XT

Common slipper shell,Crepidula fornicata[138]

DHEA ? Ad,<0.1% 17-P ? Ad,<0.1% A ? T, 20%;T ? Ad, 5%

No conversion of Ad to anyestrogens

Male-phase ovotestis(teased tissue); PC, DF,XT

Common whelk,Buccinum undatum[139]

P ? 17-P, Ad and T,some, but ‘very low’(?%)

Hepatopancreas; TLC,PC, MC (failed XT)

Sea hare, Aplysiadepilans [33,34]

Preg ? P, yes (?%) Preg ? DHEA, yes(?%); P ? 17-P andAd, yes (?%)

Preg and P ? T,yes (?%)

Gonads andhepatopancreas(acetone-driedpowder); TLC, XT

Cuttlefish, Sepiaofficinalis [40]

Preg ? P 17-Preg ? 17-P; 17-DHEA ? Ad, 0.5%

P ? 17-P, 0.5%; 17-Preg andPreg ? DHEA, 4–10%

Ad ? T, 1.5% T ? E2, 3% (ovaries only) Testes and ovaries(teased tissue); PC,TLC, DF, XT

Edible snail, Helixpomatia [47]

Preg ? P, 2–4% byovotestis; 0.1–0.6% bydorsal body; DHEA ? A,2–6%

Ovotestis(homogenate); TLC,DF, XT

Great pond snail,Lymnaea stagnalis[48]

Preg ? P, 7–14% P ? 17-P or Ad, noconversion

Ovotestis(homogenate of frozentissue); TLC, DF, XT

Garden snail, Helixaspersa [82]

T ? Ad, > 50%,(predominant injuveniles)

Ad ? E3 (but not E1 or E2),0.8%

Ad ? 5a-reducedandrogens,>30%(predominant inadults)

Gonads (homogenateof frozen tissue); TLC,DF, XT

Common periwinkleLittorina littorea[140]

Ad ? E1 and E2,<0.01% T ? 5a-DHT, ‘goodyields’

Hepatopancreasincluding gonads(microsomes);TLC

Sea slug, Clioneantarctica [30]

P ? 17-P or Ad, noconversion

Ad ? T, noconversion

No conversion of Ad to anyestrogens

P ? P(5a) and 3a-P(5a), 53%;Ad ? A(5a) and 3a-A(5a), 45%

Whole animals; TLC,DF, XT

Scallop Patinopectenyessoensis [50]

[1b-3H]Ad ? H2O. 3.2%

Grooved carpet shell,Ruditapes decussates[121]

T ? Ad, ?%(dominantmetabolite)

T ? E1 and E2, ?% T ? 5a-reducedandrogens, ?%

Hepatopancreas(microsome prep. offrozen tissue); TLC

Mud snail, Ilyanassaobsoleta [141]

T ? 5a-DHT, ?% Intact animals TLC

Purple dye murex,Bolinus brandaris[130]

[1b-3H]Ad ? H2O, activitydetected

Pacific oyster,Crassostrea gigas[55]

Ad ? steroids with samemobility as E1 and E2 onTLC, <0.1%;+ve; [1b-3H]Ad ? H2O

Gonads (homogenate)HPLC, TLC

Eastern oyster,Crassostrea virginica

T ? Ad; E2 ? E1 Sperm PC, DF, XT

(continued on next page)

A.P. Scott / Steroids 77 (2012) 1450–1468 1457

Table 2 (continued)

Species D5-3b-HSD 17-Hydroxylaseplus 17,20-lyase

17b-HSD Aromatase (CYP 19) Other enzymes Methodology

[74]

Giant ramshorn snail,Marisa cornuarietis[142,143]

Ad ? T; T ? Ad Ad ? 5a-DHA and 5a-DHT

Gonad/hepatopancreas(microsomepreparation); TLC,HPLC, GC–MS

Banded dye-murex,Hexaplex trunculus[144]

Ad ? T, 8% Gonad/hepatopancreas(microsomes);HPLC

Purple dye-murex,Bolinus brandaris[144]

Ad ? T, 10% infemales, 0% inmales)

Ad ? 5a-reducedandrogens, 80% infemales and 98% inmales

NB, conversions in bold type indicate those with ‘appreciable’ (>2%) yields.Abbreviations used: 17-P, 17-hydroxyprogesterone, 17-hydroxypregn-4-ene-3,20-dione; 20b-P, 20b-hydroxypregn-4-ene-3-one; 3a,17b-A(5a), 5a-androstane-3a,17b-diol;3a-A(5a), 3a-hydroxy-5a-androstan-17-one; 3a-P(5a), 3a-hydroxy-5a-pregnan-20-one; A, androstenedione, androst-4-ene-3,17-dione; DF, Derivative formation (micro-chemistry); DHEA, dihydroepiandrosterone, 3b-hydroxyandrost-5-en-17-one; E1, 17b-estradiol, (17b)-estra-1,3,5(10)-triene-3,17-diol; E2, estrone, 3-hydroxyestra-1,3,5(10)-trien-17-one; E3, estriol, estra-1,3,5(10)-triene-3,16 [a], 17 [b]-triol; HPLC, high performance liquid chromatography; P(5a), 5a-pregnane-3,20-dione; P, proges-terone, pregn-4-ene-3,17-dione; PC, paper chromatography; Preg, pregnenolone, 3b-hydroxypregn-5-en-20-one; T, testosterone, 17b-hydroxyandrost-4-en-3-one; TLC, thinlayer chromatography; XT, re-crystallization to constant specific activity.

Fig. 2. Basic structure of cholesterol, with the positions of the carbon atomsnumbered. The same numbering system is used for all the vertebrate steroids.

1458 A.P. Scott / Steroids 77 (2012) 1450–1468

explanation of why M. edulis failed to convert cholesterol to preg-nenolone [42,43] or why S. officinalis [40] apparently did so, butwith very low yields (<0.01%). However, a more likely explanationis that this enzyme does not exist in mollusks. Despite a report thatmRNA extracted from Mytilus spp. was able to hybridize with aprobe based on human CYP11A1 [44] and another report that anantibody against rat CYP11A1 was able to bind a protein in a par-ticular cell-type of the digestive gland of the mussel, Mytilus gallo-provincialis [45], genomic studies suggest that the gene forCYP11A1 only evolved in the vertebrate line [46]. It is of coursepossible that some other enzyme may have subsumed the role ofCYP11A1 in mollusks. However, one has to bear in mind that thegenes for most of the other critical events in steroid synthesis alsoappear to be missing [46] from the genomes of invertebrates thatare not in the direct vertebrate line. Thus what are the chances thata substitute for CYP11A1 would evolve in mollusks if they did nothave the enzymes to further transform Preg into P, T and eventu-ally E2 (unless, although unlikely, a whole suite of enzymes to per-form these activities evolved independently)?

5.7. D5-3b-HSD activity (column 2 in Table 2)

Cholesterol is unsaturated (i.e. there is a carbon double bond)between C-5 and C-6 (Fig. 2). It also has a hydroxyl group at posi-tion C-3. The four-ring skeleton is unaffected when cholesterol isconverted to Preg. However, when Preg is converted to P (or dehy-droepiandrosterone [DHEA] to Ad), the hydroxyl group is con-verted to an oxo group (by removal of two hydrogen atoms) andthe position of unsaturation moves to between C-4 and C-5; form-ing what are often referred to as ‘D4-3-one’ (or 4-pregnene) ste-roids. Most of the vertebrate steroids that are used as hormoneshave this configuration – such as P, T, KT, 17,20b-dihydroxy-pregn-4-en-3-one (17,20b-P), cortisol and corticosterone. Twostudies on two different mollusk species [47,48] have reportedrelatively high levels of this enzyme activity. Six other studies(see Table 2), however, have reported very low or no activity. D5-3b-HSD activity been histochemically demonstrated in tissues ofmaturing Pacific oyster, Crassostrea gigas (specifically in what aredescribed as ‘elongated epithelioid tissue adjacent to adductormuscle and visceral ganglia’ [49]; and in isolated cells of the testisof O. vulgaris [9]. Scattered single cells of the ovaries of the scallop,Pecten yessoensis, have also been immunostained with an antibody

against the vertebrate enzyme [50]. Since in situ staining proce-dures can never be 100% specific (plus, in all cases, the data pro-vided are qualitative), this type of evidence cannot be acceptedas firm proof of the specific presence of this enzyme.

5.8. C17,20-lyase (column 3 in Table 2)

There are in fact two key events associated with this enzyme.The first involves the insertion of an oxygen at the C-17 positionof P or Preg to form 17-P and 17-hydroxypregnenolone (17-Preg),respectively (Fig. 1); and the second involves the removal of theside chain (C-20 and C-21) to form Ad or DHEA, respectively. Theinsertion of an oxygen atom on the C-17 is an essential step, as thisatom will become the 17-oxo group of Ad or DHEA. Without anoxygen atom, the side-chain cannot be cleaved to form an andro-gen (either by an enzyme or by chemical means). It has for a longtime been thought that there was only one enzyme in vertebratesthat performed these combined actions, and that the formation of17-hydroxylated C21 steroids such as cortisol and 17,20b-P wasdue to selective inhibition of the lyase activity. However, an en-zyme has recently been characterized, from a fish, that specifically

A.P. Scott / Steroids 77 (2012) 1450–1468 1459

carries out 17-hydroxylation of P without any subsequent cleavageto form Ad [51]. It is likely that it is this enzyme that is operativewhen vertebrates make C21 hormones such as cortisol and17,20b-P. For the purposes of this review, evidence for the forma-tion of 17-P and 17-Preg is grouped with evidence for the forma-tion of Ad and DHEA from either P, 17-P, Preg or 17-Preg). Thereis only one study in mollusks [40] that has reported good yieldsfor this activity. The other nine papers (column 3 in Table 2) havereported very low or negative yields.

5.9. 17b-hydroxysteroid dehydrogenase (17b-HSD) activity (column 4in Table 2)

Although the evidence for the existence of D5-3b-HSD, 17-hydroxylase and C17,20-lyase activity in molluscan tissues is weak,the existence of 17b-HSD activity (i.e. the conversion of Ad ? T andestrone [E1] ? E2, or vice versa) is very strong (13 studies). Theyields are high, and the observations have been repeated and inde-pendently verified in a few mollusk species. The existence of thisactivity, however, does not necessarily mean that mollusks makevertebrate steroids, or that the enzyme is the same enzyme as usedby vertebrates. Such transformations (that are assisted by the co-enzyme nicotinamide Adenine dinucleotide) could be (and proba-bly are) incidental. The reaction is a relatively simple oxidoreduc-tion reaction (either adding two hydrogens or removing twohydrogens from an exposed oxo group) similar to the conversionof pyruvate to lactate (and vice versa) and there are many such en-zymes that catalyze this type of reaction in many biochemicalpathways in both the animal and plant kingdoms [52]. HSDs de-rived from bacteria, for example, are used widely in the laboratoryas reagents for steroid conversions, such as preparing 17,20b-Pfrom 17-P [53].

5.10. 5a-reductase (column 6 in Table 2)

Quite a few mollusks (nine studies) appear able to reduce thedouble bond between C-4 and C-5 of Ad, T or P and convert themto 5a-reduced steroids (see also [28]). Although 5a-reduction ofthe A ring occurs in many vertebrates (and is important in the hu-man male for the production DHT from T), it does not only affectsteroids. The bile acid-derived compounds that lampreys use aspheromones are also 5a-reduced [54] and these are ‘sterols’ as op-posed to ‘steroids’. Like the presence of 17b-HSD activity, the pres-ence of 5a-reductase activity does not in any way imply thatmollusks make vertebrate steroids, nor that such steroids are theirnormal substrate.

5.11. CYP19 (aromatase; column 5 in Table 2)

E2 is made from T, and E1 from Ad, by a rather complex reactionthat involves an enzyme called aromatase. There are two ways todemonstrate aromatase activity in tissues. One is to add radiola-beled Ad or T to the tissues and then establish whether they havebeen transformed to radiolabeled E1 and/or E2. The other is to addT that has been labeled with tritium at the C-1 position and then tomeasure how much is converted into water (‘released’) during thereaction. Using the first procedure, three studies found no conver-sion of androgens to estrogens, three found <0.01% conversion, onefound 3% conversion, and one found apparent conversion of Ad toestriol but not to E1 or E2 (see Table 2 for references). All ‘tritiumrelease’ assays have given positive signals that have been describedas close to background measurements [28]. In C. gigas, the aroma-tase activity was noted to be only 0.1% of that found in a similaramount of bovine tissue [55]. With most studies suggesting thataromatase activity is either absent or very low, one wonders how

specific the activity is (i.e. could the weakly positive results haveother explanations such as misidentification of the products, con-tamination and actions of unrelated metabolic enzymes?). Usingantibodies against mammalian CYP19, immunohistological stain-ing has been demonstrated in scattered single cells of the ovariesof P. yessoensis [50] and in scattered cells of the testis of the samespecies [56]. However, non-specific cross-reactivity cannot be ru-led out. The most powerful argument against the presence ofCYP19 in mollusks is that the gene only first appeared in a directancestor of the chordates [57,58].

5.12. A strong streak of anthropomorphism in mollusk studies

As pointed out by Markov and colleagues [46], all the biosyn-thetic studies that have been carried out in mollusks have lookedspecifically for transformations that are essentially part of the hu-man steroid biosynthetic pathway (i.e. asking ‘can Preg be con-verted to P; can P be converted to 17-P; can 17-P be converted toAd; can Ad be converted to T; and can T be converted to E2?’).Any other products of the incubations have (with a few exceptions)been ignored. This is perhaps not surprising, as it is rather difficultand expensive to identify a steroid for which one does not alreadyhave some sort of an idea of what it might be. The normal identi-fication procedures for steroids (thin layer chromatography, micro-chemical conversion and recrystallization to constant specificactivity) all rely on having synthetic standard steroids with whichto compare the radioactive steroids that are formed in the incuba-tions. Unsurprisingly, those that are commercially available are al-most all variants of known vertebrate steroids. An example of whathappens when an incubation does not generate immediately rec-ognizable steroids is illustrated by the study on P. magellanicus[31]. In some experiments within this study, the gonads and hepa-topancreas of this animal converted up to 70% of added radiola-beled Preg and 17-P into several metabolites. However, DavidIdler, who was arguably the greatest steroid chemist of his time(he discovered both 17,20b-P and 11-KT in teleost fish) was unableto identify a single one of these (apart from a trace of Ad)!

The same criticism of latent anthropomorphism also applies tostudies in which steroids have been measured in mollusks (Table 1).The vast majority of studies appear to have been concerned withconcentrations of what are essentially human steroids (i.e. T, E2and/or P). Why not 11-KT and 17,20b-P, that are, respectively,the main androgen and the main progestin in teleosts [59]? Orwhy not 15a-hydroxprogesterone that is the dominant progestinin the blood plasma of mature males of the sea lamprey, Petromy-zon marinus [60]? And when measuring vertebrate stress steroids[17], why choose cortisol (the human stress steroid) rather thancorticosterone (which is the stress steroid in many other verte-brates as diverse as rodents and amphibians)?

5.13. Interim conclusion

It is clear that mollusks are able to carry out transformations onvertebrate steroids. However there is no solid evidence that any ofthese transformations indicate the presence of a pathway involvedin the formation of vertebrate-type steroids; nor indeed of path-ways involved in the formation of ‘mollusk-type’ steroids. Thetransformations can all be interpreted as ‘metabolism’ – thoughwhether any of the transformations are ‘deliberate’ (i.e. part of apathway specifically geared to the metabolism of vertebrate ste-roids of exogenous origin) or ‘incidental’ (i.e. the steroids are actedon non-specifically by general metabolic enzymes) is as yetuncertain.

1460 A.P. Scott / Steroids 77 (2012) 1450–1468

6. The animals pick up the steroids from the environment?

6.1. Possible sources of steroids in the environment

If one accepts the evidence that T, E2 and P definitely are pres-ent in mollusks, then the only other possible source is from theenvironment (i.e. water, sediment and food). Two major inputs ofvertebrate steroids into the environment (especially in rivers) aresewage treatments works [61] and farm animal effluents [62].However, any free-living wild vertebrate is a potential source ofsteroids. For example, in fish, there is clear evidence that there iscontinuous traffic of steroids across the gills – as well as frequentrelease via the urine and feces [63–65]. Overall, the majority of ver-tebrate steroids probably find their way into the environment viaurine and feces. Although, when they are excreted via these routes,they are mainly present as ‘biologically inactive’ glucuronide orsulfate conjugates, many organisms appear to contain enzymesthat can readily convert them back into ‘free’ steroids. Of particularrelevance to this review, one of the most abundant sources of suchenzymes (which are frequently used in the laboratory for hydroly-sis of vertebrate steroid conjugates) is mollusks! Preparationsmade from the hepatopancreas of the edible snail, Helix pomatiaand the keyhole limpet, Patella vulgata, are sold commercially bySigma–Aldrich Inc. under the headings of ‘glucuronidase’ and ‘sul-fatase’. In other words, even if the steroids in the environment arepresent as conjugates, some, possibly all, mollusks have a strongcapability of converting them back to free steroids. Bacteria in sew-age treatment works are also known to convert glucuronidatedestrogens back into their free and biologically active forms [66].

Since every vertebrate is a fairly continuous emitter of steroids(whether free or conjugated), it is unlikely that there are manyparts of the aquatic environment which do not contain some ver-tebrate steroids (whether free or conjugated). This point has beenmade previously in relation to chemical pollution [1] i.e. that thereis probably nowhere on earth that has not been affected by hu-mans. Essentially, any statement to the effect that ‘the animalswere collected from an unpolluted site’ can probably be describedas ‘wishful thinking’.

Fig. 3. The mechanism of the esterification reaction. The steroid atoms are shownin bold type. Note that the steroid must have a hydroxyl (also known as an ‘alcohol’group) in order to participate in the reaction. Also note that some steroid hydroxylgroups cannot participate in conjugation reactions at all, due to steric hindrance(e.g. that attached to C-17 of 17-hydroxypregn-4-ene-3,20-dione) and, for others,there might not be an appropriate enzyme (e.g. the C-3 hydroxyl group of 17b-estradiol and estrone in mollusks [76]).

6.2. Experimental evidence that mollusks can take up and storeexogenous vertebrate steroids

In laboratory experiments, it has been shown (and indepen-dently verified) that mollusks have a remarkable ability to absorband retain at least two vertebrate steroids (namely T and E2). Fur-thermore it has been discovered [67] that a key part of the mech-anism that allows them to retain these steroids is by conjugatingthem to fatty acids (by a process referred to as ‘esterification’;Fig. 3). The initial study on the eastern mud snail, Ilynassa obsoleta[67] showed that it took one animal only eight hours to remove80% of 14C-labeled T from 3 ml solution. When the animal wastransferred to a clean solution, hardly any of the label was releasedover 90 min. The activity was found to be associated with apolar(i.e. lipid-like) compounds. It was further shown that these com-pounds could be made in vitro and that their production could beenhanced by addition of palmitoyl coenzyme A. Subsequentstudies [68,69] showed that exposure of I. obsoleta to T via injectionor water had hardly any effect on free T levels, but had a dose-dependent effect on concentrations present as fatty acid esters.

Other studies that have demonstrated the ability of mollusks toabsorb steroids have been made using: C. gigas [70], in which indi-viduals were shown to bioaccumulate 14C-E2 from water, with abioaccumulation index (the ratio of activity in 1 g tissue to activityin 1 ml water) of 30 after 48 h; M. edulis [71], in which individualsthat had been exposed to 200 ng L�1 of E2 for 10 days increased

their levels of free E2 from 0.3 to 2.5 ng g�1 and of esterified E2from <1 to 17 ng g�1; the zebra mussel, Dreissena polymorpha[72], in which 14C-E2 that had been added to the water was shownto bioaccumulate over a period of 13 days by a factor of 840 inmales and 580 in females, and when the animals were transferredto freshwater, the activity was retained (with no apparent loss ofactivity) for at least 10 days, and also, when the tissues were finallyextracted, the esterified E2 was found not to have been metabo-lized in any way; M. galloprovincalis [73] in which it was shownthat, when T was added to water for 5 days, it was taken up in sub-stantial amounts – and esterified T increased in a dose-dependentmanner, while the levels of free steroid were relatively unaffected;the eastern oyster, Crassostrea virginica [74] and the Giant rams-horn (Apple) snail, Marisa cornuarietis [75], in which it was shownthat levels of esterified (but not of free) E2 also increased in a dose-dependent manner; M. edulis again [76], in which 14C-E2 wasshown to reach a bioconcentration factor of 2500 after 13 daysand have a half-life of depuration of 8.6 days, and all the activitywas all associated with a single peak on HPLC that, after basehydrolysis, was reconverted to pure E2. In this last study, whenthe animals were exposed to 14C-E1, they also readily absorbed thissteroid (with a bioconcentration factor of 1000 after 8 days). How-ever, when the esters were subjected to base hydrolysis, the ste-roid that was released was 14C-E2.

The above-mentioned studies on D. polymorpha [72] and M.edulis [76] are particularly interesting, because, in the first case,the authors found from sampling of animals in the wild, thatalthough the sediment in which the animals lived contained farmore E1 than E2, only esterified E2 could be detected in tissues;and, in the second case, the authors found, as just mentioned, thatif they added 14C-E1, it was converted to and then stored as 14C-E2.These results indicate that there is something about E1 that pre-vents it being conjugated to fatty acids. The only difference be-tween E1 and E2 is that the latter has a C-17b-hydroxyl grouprather than a C-17-oxo group. In order for a fatty acid to be com-bined with a steroid, a molecule of water must be formed and itis a pre-requisite that the steroid has a hydrogen atom to donateto the reaction (Fig. 3). Because the @O group on position 17 ofE1 does not have a hydrogen atom, conjugation is impossible. Ste-roids such as P and Ad that do not have any hydroxyl groups at allcan also not be conjugated. In theory, E1 could be conjugated via its3-hydroxyl group. However, this is an unusual group in that it isattached to an aromatic ring; and the fact that no conjugated E1was found in the study on M. edulis indicates that it does not takepart in conjugation reactions, in this species at least. Other steroidhydroxyl groups that are known to be unreactive to conjugatingenzymes in vertebrates (and the same would probably be the casein mollusks) are those at position C-11b of cortisol and at positionC-17 of 17-P. However, the C-3b-hydroxyl group of DHEA is onethat is reactive in mollusks, as this steroid was readily esterifiedin vitro by microsome preparations of digestive glands and gonadsof C. virginica [74]. Incidentally, in M. cornuarietis [75] it was shown

A.P. Scott / Steroids 77 (2012) 1450–1468 1461

that rates of esterification of E2 and T were correlated, supportingthe existence of a single enzyme that in effect works on an identi-cal C-17b-hydroxyl group in both steroids.

Working on the basis that T that might be responsible for stimu-lating penis development in female mollusks (a hypothesis ex-plained in detail by Matthiessen and Gibbs [77]), severalinvestigators have looked at whether the way in which TBT exertsits effect on penis formation is perhaps by inhibition of the esterifi-cation of T – thereby leading to an increase in the levels of free T.However, most studies, either in vivo [68,75,78] or in vitro [79] showonly small effects of TBT on esterification. Only one in vitro study onI. obsoleta [80] reported relatively pronounced effects of TBT.

There is further (albeit circumstantial) evidence from field stud-ies that indicates that mollusks tend to pick up steroids from theenvironment. For example, in P. antipodarum that had been cagedin rivers, steroid concentrations in individuals always increasedwith time, irrespective of the external conditions. There was a 5-fold increase in total (i.e. free and esterified) amounts of T and Pin animals caged downstream of a STW for 21 days [15]. In anotherstudy [14], animals were also placed in cages upstream and down-stream of STWs and total levels of E2, T and P again increasedmarkedly (5- to 10-fold) with time at all downstream sites (andto a lesser extent upstream). The same increase with time, but withno link to the conditions being tested (in that case metal contam-ination), was noted in yet a third study by the same group [11].Although these increases could possibly have been due to stimula-tion of endogenous steroid synthesis by unknown factors in theriver water, the only common factor in all the experiments was‘time of exposure’.

6.3. What is the point of esterification?

The question everyone who works in this field has not yet beenable to answer is ‘what is the relevance of conjugation of T and E2 tofatty acids by mollusks?’. These two steroids are known to be avail-able exogenously in the environment and both possess the samereactive C-17b-hydroxyl group. For all we know at the moment,they may just be two among many other compounds with hydroxylgroups that are capable of being linked to fatty acid groups; and weonly know about these two compounds because they are the onlyones that anyone has so far been interested in measuring. In fact,in the pioneering study on I. obsoleta [67], it was shown that, afterbase hydrolysis of apolar material extracted from the animals,although T was identified among the products, there was a large ex-cess of free fatty acid (indicating that there were esters of othercompounds in the total mix). If one could get some handle on theidentity of the other compounds that are stored as esters in mol-lusks, it might give some clue to the function of this mechanism.

6.4. What can explain the presence of steroids in laboratory-rearedanimals?

Those researchers that have published the clearest evidence forthe uptake of vertebrate steroids from the water [10,72,76] freelyacknowledge the likely impact of uptake on steroid concentrationsin molluscan tissues, but appear to be reluctant to abandon theconcept that the same steroids are also produced endogenously.In order to defend the endogenous origin of steroids in animals keptin the laboratory, arguments are used such as: even though col-lected in the wild, the animals had been kept in the laboratory forseveral months in artificial sea- or fresh-water [81]; the animalshad been hatched and grown entirely under clean laboratory condi-tions [75]; or the species was land-living [17,82]. Essentially, wherecould the steroids have come from if the water was clean (or therewas no water) and there were thus no upstream effluents or fish toworry about? The answer may lie (it is suggested) in the fact that

the animals have to be maintained by humans. Humans are notinconsiderable synthesizers of P, T and E2 – the three steroids thathave been mainly studied in mollusks. It is well known that humans(like all vertebrates) excrete steroids via the urine and feces. How-ever it is not so well known that they also excrete steroids via saliva,sweat and skin [83–85]. Even if mollusks (and their food) are at alltimes handled with gloves, it still has to be taken into account thathumans are continuously shedding their skin and hairs. In fact, hu-man ‘skin scales’ (potentially contaminated with steroids) are a ma-jor constituent of airborne dust in most buildings [86]. Even thoughthe amounts of dust and associated steroids entering rearing tanksmight be relatively low, the proven ability of mollusks to bioaccu-mulate vertebrate steroids would probably ensure a build up ofmeasurable quantities over the many months that mollusk test spe-cies are normally kept in the laboratory. Another obvious potentialsource of steroids for mollusks is their food – especially if the foodtakes the form of ‘organic lettuce’ that may well have been fertilizedwith animal (vertebrate) manure.

As confirmation that steroids are present on human skin, volun-teers at the Centre for Environment, Fisheries and Aquaculture Sci-ence were asked to dip one of their arms (up to the elbow) into aplastic bag containing either 1 L or 1.5 L of water for 1 min. Thewater was then extracted and assayed for cortisol, T and E2. Inthe first trial with 29 participants, the amount of cortisol extractedfrom the 1 min water samples ranged from 2000 to 120,000 pg andT ranged from 800 to 6000 pg. In the second trial, with 38 partici-pants, E2 ranged from 80 to 540 pg, T from 580 to 3400 pg, P from1500 to 4900 pg and cortisol from 3400 to 32,000 pg. The datafrom these trials are still being analyzed (and thus only reportedhere in summary). They are mentioned ahead of full publicationpurely to make the point that the steroids that we make are notconfined within our bodies or totally excreted via either our urineor feces. Some of these steroids appear on our body surfaces – andwe need to take this into account when thinking about likelysources of contamination in the laboratory.

The hypothesis outlined above that mollusks pick up human/mammalian steroids as a result of steroid contamination obviouslyrelies upon those steroids being relatively stable in the environ-ment. In fact, in comparison to many other types of organic com-pounds that are handled in the laboratory (e.g. peptides andprostaglandins), steroids are very stable indeed. In a paper that isquoted as evidence that algae make vertebrate steroids [87], themost impressive fact is not the identity of any of the metabolites(there was, incidentally, no evidence of any 17-hydroxylation orformation of androgens), but the fact that in all 12 species thatwere examined, the bulk of the P that was added as a precursorwas recovered intact (i.e. not broken down or metabolized in anyway) after having been stirred in an aqueous solution, underlighted conditions for 14 days at 24 �C!

Having said all the above, it has to be stressed that there is, at themoment, no actual proof of steroid transfer between humans andlaboratory stocks of mollusks. It is purely a hypothesis. However,as a potential explanation for the presence of human steroids inmollusks, it has more plausibility than the hypothesis that molluskshave evolved their own alternative biosynthetic pathway for mak-ing human-like steroids. Whatever the merits of either hypothesis,until cross-contamination has been excluded experimentally in thelaboratory, it cannot be assumed that the presence of steroids insupposedly ‘clean’ laboratory stock animals necessarily means thatthe animals must have been able to make the steroids themselves.

6.5. Looking for steroids that are unlikely to have been formedendogenously

One possible way to examine the problem of potential cross-contamination in laboratory stocks would be to look for the pres-

1462 A.P. Scott / Steroids 77 (2012) 1450–1468

ence of steroids that one would not expect the mollusks to be ableto make themselves. A potential candidate would be cortisol (that,as shown above, is found in substantial levels on human skin).However, it would first need to be shown that mollusks have theability to bioconcentrate and accumulate this steroid in the sameway as they do with T and E2. Another candidate would be EE2(which is a part of ‘the pill’ and does not occur in nature at all).In fact, there are two studies that have already convincingly dem-onstrated the presence of EE2 in the flesh of wild-caught mollusks[7,88]. In the second of these studies, five mollusk species (includ-ing the abalone, Haliotis diversicolor supertexta) from a bay in Chinawere analyzed and, while E2 levels were below the levels of detec-tion, EE2 levels were an astonishingly high 80,000–130,000 pg g�1

of dry weight of EE2 in all five species. Subject to independentverification, these findings are, at the very least, strong positiveevidence that at least some (if not all) of the steroids that havebeen measured in mollusks are of exogenous origin.

7. The evidence for steroid receptors in mollusks

7.1. Presence of classical nuclear receptors

Most, if not all, studies carried out on steroid receptors in mol-lusks to date (Table 3) have been based on the assumption that, ifsuch receptors exist, they are so-called ‘classical’ nuclear receptors.These are proteins that have a characteristic structure – a highlyconserved DNA-binding domain and a moderately conserved li-gand-binding domain – that makes them easy to recognize withinthe genomes of animals; and analysis of proteins with such struc-tures in the genomes of a variety of animals [89] suggests thatthere were 25 ancestral nuclear receptors in Urbilateria (the hypo-thetical ancestor of all animals with bilateral symmetry). These 25receptors have diversified (variously being replicated and lost) inthe animal kingdom to serve as mediators for a large array of dif-ferent compounds including vertebrate steroids. In relation to ste-roids, the present thinking is that one of these ancestral nuclearreceptors was and always has been a receptor that was able to bindto estrogens [90,91]. The ‘classical’ nuclear receptors for proges-tins, androgens and other vertebrate steroids, however, are be-lieved to evolved from the nER only after the divergence of thevertebrates [57,92].

If this scenario is correct, then one would expect to find thegene for nER in mollusks, but no genes for nuclear progestin recep-tor (nPR) or nuclear androgen receptor (nAR). The facts do indeedappear to match the expectation. There is a gene in all mollusksthat have so far been studies that is the undoubted analogue of ver-tebrate nERs (publications listed in Table 3) and no-one has yetidentified any genes that match nuclear receptors for other verte-brate steroids. However, despite the presence of nER-like proteinsin mollusks, the ligand-binding domains appear to be unable tobind to estrogens (i.e. they are structurally like nERs but are notfunctional nERs) [93,94]. The mollusk nER still binds to DNA andactivates genes, but because it does this without the need for anyligand at all, it has been termed a ‘constitutive receptor’. The theorythat the ancestral steroid receptor was an ‘estrogen’ receptor hasgained support from the demonstration that the earthworm (notin the line of evolution of vertebrates or mollusks) has an nER thatis able to bind to estrogens [90]. In other words, the nER in mol-lusks is more likely to have ‘lost its ability to recognize estrogens’rather than ‘not evolved the ability to recognize estrogens’.

Alert readers will almost certainly have picked on an anomaly.The author has argued above that there is no firm evidence (only‘poor’ and at best ‘circumstantial’ evidence) that mollusks biosyn-thesize vertebrate steroids. Furthermore, the genomic evidenceshows that two critical enzymes necessary to make estrogens,

aromatase (CYP19) and the cholesterol side chain cleavage enzyme(CYP11A1) only evolved in the immediate ancestors of the verte-brates [46,58]. How come then that the ancestral steroid nuclearreceptor was an estrogen receptor? This anomaly has already beennoted by Baker [95,96], who has suggested that, while the primitivenER may well have been able to bind to estrogens, it probably, at thattime in evolution, had a ligand that was not actually E2 (as it is to-day). This is entirely plausible. One interesting fact about the verte-brate nER (of which the human b form has received the mostattention) is its relative promiscuity compared to the other steroidnuclear receptors. There are estimated to be hundreds, probablythousands, of compounds in the environment (both natural andman-made) that are able to activate the human nERb (admittedlymostly with many times less potency than E2). Such compounds(examples are alkyphenols, BPA, genistein, some DDT metabolites)are very diverse in their structure and most of them look nothing liketypical estrogens [97,98]. Quoting from Barnes [97]: ‘in general, pla-nar compounds with a phenolic character and two oxygen-containingmoieties approximately 11–12 Å apart can fit into the bindingpocket’ of the nER. Thus while Baker has suggested (see above) thatthe primitive ligand might have been an androgen or a sterol derivedfrom cholesterol, the present author suggests that it might not evenhave been a steroid at all. However, this is pure speculation.

This brief (and rather oversimplified) potted history of the evo-lution of nuclear steroid receptors does have important implica-tions for the interpretation of receptor studies in mollusks. Thereare a few studies that have reported the presence of steroid bind-ing in mollusk tissues (Table 3). Also, in some of these studies, thebinding appears to do everything that a classical nuclear receptorwould be expected to do [99,100], including binding to DNA. How-ever, if the genomic studies are to be believed, this cannot be so –because the nAR and nPR should not be present in mollusks andthe nER is unresponsive to estrogens. So what explanations couldthere be?

7.2. Another class of nuclear receptor?

This is a distinct possibility. In humans, a different ancestral nu-clear receptor lines has given rise to a protein that is able to bind awide range of compounds including progestins, corticosteroids,bile acids and E2 [101]. In response to these compounds, this bind-ing protein, called the Pregnane X Receptor, activates genes specif-ically involved in their metabolism and excretion (i.e. as isdiscussed in Section 7.5, it appears to have a role as a ‘sensor’rather than a ‘receptor’).

The ability of a binding protein to bind to DNA–cellulose is usu-ally accepted as proof that that particular protein is a nuclearreceptor [102]. Since the binding proteins for P and E2 in O. vulgarisbind to DNA–cellulose [99,100], this does indeed suggest that theremay be another class of nuclear receptors in mollusks that are ableto bind steroids. However, one must probably be a bit cautious inthe interpretation of such data. The procedure for characterizingbinding proteins in tissues is very crude. The tissues are essentiallymixed with a buffer and then turned into a ‘cold soup’ that is thenseparated, purely by centrifugation steps, into what are termed the‘cytosolic’, membrane’ and ‘nuclear’ fractions. It is these relativelycrude fractions that are tested for binding and put throughDNA–cellulose columns. As one can imagine, the binding proteinsform a tiny proportion of the total organic content of these fractions,and who really knows what interactions there might be with othercompounds that might give the appearance of a protein being ableto specifically bind to DNA. Another problem that one must watchout for is that a binding protein might have the appearance of beingsoluble (i.e. it is present in the cytosol fraction), but on closerexamination, turns out to be ‘membrane-bound’. This was foundto be the case for the putative ‘androgen receptor’ of the sea

Table 3Evidence for presence of steroid receptors in mollusks.

Species (author) Hormone Type of evidence (and dataprovided)

Location and approximateamounts

Other findings Comments

Giant ramshornsnail, Marisacornuarietis[145]

E2 Molecular biology: Cloningof protein similar toclassical vertebrate nER

In all tissues tested, but slightlymore in penis and sheath (not highin gonads)

Zero binding to E2

Giant ramshornsnail, Marisacornuarietis[75]

E2 and T Binding activity: specificityonly

Testis and ovary cytosols Testis and ovary had similar binding

Dogwhelk, Nucellalapillus [146]

E2 Molecular biology: Cloningof protein similar toclassical vertebrate nER

3-fold increase in expression inovary after exposure to 0.25% (butnot 1%) effluent

They comment: structuralsimilarity to Thais clavigera nERsuggest it does not bind E2

Eastern mudsnail,Ilyanassaobsoleta [119]

E2, T Molecular biology: Cloningof protein similar toclassical vertebrate nER

Gonad-viscera complex Changes (up to 5-fold) noted by timeof year and reproductive stage

No gene similar to vertebratenAR

Freshwatermussel, Elliptiocomplanata[147]

E2 Immunoreactive bands onPAGE using human anti-nERb

Three bands (55, 75 and 165 kDa)in foot, hepatopancreas and gonads

Freshwatermussel, Elliptiocomplanata[148,149]

E2 Binding activity: affinity,capacity, specificity

Testis and ovary cytosol Less at one contaminated site thantwo others

Used E2-BSA-fluorescein

Marine snail, Thaisclavigera [94]

E2 Molecular biology: Cloningof protein similar toclassical vertebrate nER

Four times more in ovary andtestis; also present in ganglia

Zero binding to E2

Mussel, Mytilusedulis [104]

E2 Immunoreactive bands onPAGE using human anti-nER3a and anti-nERb

Hemocytes Involvement of nuclear receptorsin mediating rapid effects notdiscussed

Mussel, Mytilusedulis [150]

E2 Molecular biology: PCRquantification of musselnER mRNA

Gonads Recorded large changes in responseto estrogen exposure (moreresponsive in Feb than Apr)

Not dose-responsive to E2; cf.negative response to estrogens in[151]

Mussel, Mytilusedulis [151]

E2 Molecular biology: Cloningof protein similar toclassical vertebrate nER

Gonads Not affected by exposure to E2 forten days

Mussel, Mytilusedulis [152]

E2 Immunoreactive bands onPAGE using human anti-nERb

Positive staining of proteinsextracted from membranes andcytosol of pedal ganglia

Also showed that NO release bypedal ganglia in vitro could bestimulated by E2 coupled to BSA

They suggest: there may twotypes of receptor (nER andmembrane-bound ER)

Mussel, Mytilusedulis [153]

E2 Molecular biology: Cloningof protein identical tohuman nER

Identity to human nER almostcertainly due to contamination

Octopus, Octopusvulgaris male[9]

E2, T andP

Binding activity: Affinity,capacity, specificity

Only P and T binding in testis andseminal vesicle; P, T and E2 in vasdeferens an prostate

Not much binding in non-reproductive tissues

Octopus, Octopusvulgaris female[154]

P Binding activity: Affinity,capacity, specificity, DNA-binding

Anti-chicken nPR stained band onPAGE (70 kDa) and ovarian tissues

They comment: characteristicsare those of classical vertebratenPR

Octopus, Octopusvulgaris female[99]

E2 Binding activity: Affinity,capacity, specificity, DNA-binding

In ovary and oviduct (in cytosol butnot nucleus); none inhepatopancrease or hemolymph

Anti-human nER stained band onPAGE (mass 70 kDa) and in nuclei ofovarian follicle cells

Octopus, Octopusvulgaris [93]

E2 Molecular biology: Cloningof protein similar toclassical vertebrate nER

Extracted from ovary and oviduct Zero binding to E2

Pacific oyster,Crassostreagigas [155]

E2 Molecular biology: Cloningof protein similar toclassical vertebrate nER

In all tissues tested, but most inovary, testis, labial palp and gills

Zero binding to E2; anti-oyster nERimmunostained nuclei of follicle cellsand oocytes in ovary

Scallop,Placopectenmagellanicus[156]

E2 Molecular biology: Cloningof protein similar toclassical vertebrate nER

They were worried aboutcontamination (close to sequenceof rainbow trout nER)

E2 Binding activity: affinity,capacity, specificity

Testis and ovary (cytosol andnucleus)

Nuclear binding lower in spent thanripe females

They comment: less specific thanvertebrate nERs

Abbreviations used: E2, 17b-estradiol; kDa, kilodalton; nAR, ‘classical’ nuclear androgen receptor; nER, ‘classical’ nuclear estrogen receptor; nPR, ‘classical’ nuclear proges-terone receptor; P, Progesterone; T, Testosterone.

A.P. Scott / Steroids 77 (2012) 1450–1468 1463

lamprey, P. marinus [103]. In this primitive vertebrate, bindingactivity for Ad and T is present in impressive quantities in the cyto-

sol fraction of the testes. Furthermore it binds to DNA–cellulose.However, on closer examination, the activity is found to be part of

1464 A.P. Scott / Steroids 77 (2012) 1450–1468

a neutrally-buoyant lipid complex that had a mass in excess of>1000 kDa. Since the receptor is not actually dissolved, this callsinto question the relevance of binding to DNA–cellulose. Is it genu-ine or an artefact? Yet another point to bear in mind is that manypeople use properties such as ‘affinity’, ‘binding capacity’, ‘half-lifeof association’, ‘half-life of dissociation’ and ‘specificity’ in order toargue the point as to whether the binding is ‘typical’ of a nuclearreceptor. However, as discussed by Bryan and colleagues [103],there is far too much overlap between the properties of nuclearreceptors and other types of binding protein (see below) to be ableto reach any such conclusions. Only knowledge of the structure ofthe binding moiety, plus exhaustive testing of its function, can pos-sibly answer the question as to whether it is a receptor or not.

7.3. Could it be a ‘non-classical’ membrane-bound receptor?

The concept of membrane-bound steroid receptors has alreadybeen introduced. This is an ever-expanding field in vertebrates, andsome researchers have already suggested that this may be the wayin which steroids exert their effects in mollusks [27]. However, thisis at the moment speculation. Certainly, the fact that isolated cellsfrom some mollusks show very rapid responses to E2 in vitro [104]seems to imply the existence of a membrane-bound receptor thatrecognizes this steroid. However, it is unclear whether this repre-sents the presence of a specific receptor for E2, a receptor for someother compound with which it cross-reacts (e.g. [105]) or indeed,any receptor at all.

At the moment, there are three ‘main players’ as membrane-bound steroid receptors in vertebrates [106–108]. These are: themembrane progestin receptor (mPR; of which there are five vari-ants, labeled from a to c); the progesterone membrane receptorcomponent (PGMRC; of which there are two variants, labeled 1and 2); and the membrane estrogen receptor (termed GPR30).However, there are more possibilities (see [108]).

Anyone setting out to identify membrane-bound receptors inmollusks should be aware that, even within the vertebrate field,there is presently a fierce debate about whether the mPRs andPGMCRs actually are progestin receptors; and also whether GPR30really is an estrogen receptor [107,108]! It could ultimately proveunrewarding looking for homologous sequences of one or other ofthese putative membrane receptors in mollusks if one then findsout that the original protein was not a steroid receptor after all.The gene for PGMRC1, for example, is likely to be present in mollusks,as it has an ancient lineage, and its protein product has been purifiedfrom another invertebrate, the rotifer, Brachionus manjavacas [109].Although it was one of several proteins pulled out of a crude rotiferextract using a progesterone-labeled affinity reagent, its presencemay be fortuitous, because, as stated by two experts in the field ofmembrane-bound receptors [106,107] ‘it has yet to be demonstratedthat PGRMC1 exhibits specific progesterone binding’!

7.4. Could there be binding proteins that are not receptors?

In vertebrates, there are a well-known class of proteins, the ‘ste-roid-binding globulins’ (SBGs) that circulate in the plasma [110]and are also expressed in the testes (androgen-binding protein).SBGs that bind T and E2 with high affinity and specificity are foundin most vertebrates that have been studied. These proteins (thefunctions of which are still debated) have binding properties thatare very similar to those of nuclear receptors.

Albumin [111] and even egg yolk protein [112] are able to bindto steroids (the latter with very high capacity). However, these pro-teins do so with considerably lower affinities than those for nuclearreceptors or SBGs (and are thus unlikely to explain the steroidbinding activity of O. vulgaris tissue extracts, for example).

Any enzyme that is able to modify a steroid should in theory beable to bind that steroid temporarily (in order to effect the modifi-cation). However, although the present author has heard this men-tioned as a possible explanation for binding activity, he has notfound an example to quote.

7.5. Does the presence of a receptor necessarily mean the ligand has tobe endogenous?

Even if, after further research, it is found that there actually is areceptor for a steroid in mollusks, the question that then needs to beanswered: ‘is this fortuitous (i.e. the receptor evolved to mediatethe action of an entirely different compound but, by reason of shapeand size, the steroid ‘fits’ the binding site) or by design (i.e. thereceptor specifically evolved to mediate the action of that steroid)?’.If it by design, then yet a further question is ‘does that necessarilymean that the steroid has to be of endogenous origin?’. The ances-tors of protochordates and vertebrates appear to have been the firstanimals to have the capability to synthesize steroids (as opposed tosterols), and these evolved about 500 million years ago [58]. Thismeans that steroids have likely been around in the aquatic environ-ment for a considerable part of mollusk evolution. It is pure specu-lation, but if there was perhaps something about the shape of somesteroids (e.g. E2) that made them interfere (even weakly) with someother types of receptor in mollusks, then this might have providedsufficient evolutionary pressure for the development of bindingproteins that would recognize them in order to facilitate their deac-tivation. In fact, Baker [113] has hypothesized that it might havebeen the necessity to recognize and deactivate xenobiotics that en-abled the evolution of receptors; and has pointed out that somereceptors appear to have retained that role – e.g. the aryl hydrocar-bon receptor, the main ligand(s) for which appear to be toxic com-pounds that the animals have absorbed or ingested and the mainfunction of which is to activate P450 enzymes in the liver to metab-olize those compounds. In relation to E2, the only effect that has sofar been properly verified in mollusks (Mytilus spp.) is its ability tovery rapidly (<2 min) cause a decrease in lysosomal membrane sta-bility [104,114]. As pointed out by Canesi and coworkers [115], thisis a classic ‘immune response’ rather than an ‘endocrine response’,thus strengthening the hypothesis that, as far as some mollusks areconcerned, E2 is more likely to be a xenobiotic ‘inflammatory agent’rather than an endogenous hormone.

7.6. Is a receptor necessary to elicit effects?

The fact that steroids are taken up by mollusks, and are thenesterified, means that biochemical and physiological changes takeplace in the organisms, whether or not they are taken up by a sys-tem that recognizes them specifically. For all we know at the mo-ment, mollusks might just treat the steroids in the water as aconvenient source of soluble food. When steroids such as T andE2 are added to the water, the animals presumably need to mobi-lize (and probably even manufacture fatty acids). They also need toactivate the conjugating enzymes. These steps all require energyusage and re-allocation of resources. This makes it possible thatany effects that might be noted when these steroids are added tothe water might be due to these sorts of changes rather than toany receptor activation.

8. Conclusion

Despite many studies failing to take account of the possibility ofnon-specific assay interference, vertebrate-type steroids canundoubtedly be detected in molluscan tissues. Furthermore, insome studies, it has been claimed that the concentrations of these

A.P. Scott / Steroids 77 (2012) 1450–1468 1465

steroids are related to changes in the reproductive cycle, are higherin one sex than another or are modified by presumed ‘endocrinedisrupters’. However, despite studies starting over fifty years ago,no-one has come up with clear evidence that any mollusk can actu-ally make vertebrate steroids de novo (although there is little doubtthat mollusks can metabolize vertebrate steroids – e.g. saturate theA ring or convert E1 to E2). Individual steps of the biosyntheticpathway have been demonstrated in some species, but yields havein most cases been zero or very, very low (and have, furthermore,never been independently verified). Crucially, the genes for key en-zymes involved in steroid biosynthesis in vertebrates are missingfrom the genomes of mollusks. This means that, if mollusks domake T, E2 and P, they will have to do so via an independentlyevolved set of enzymes. Unless there is something remarkableabout these particular steroids for the whole animal kingdom (orindeed most life forms – because there are even claims that plantscan synthesize mammalian steroids [116], then the developmentof independent pathways making exactly the same steroidal end-products is very unlikely to have occurred.

Furthermore, there is still no firm evidence for the presence of spe-cific functional receptors for vertebrate steroids; nor firm evidencethat vertebrate steroids have endocrine effects on mollusks [4].

In contrast to the weak evidence for biosynthesis and function-ality of vertebrate steroids in mollusks, there is strong evidencethat mollusks can readily pick up vertebrate steroids from theenvironment. If the steroid has a reactive hydroxyl group (e.g.the C-17b-hydroxyl group of both T and E2), then mollusks canreadily conjugate it to a fatty acid. The resulting esters can be re-tained in the animals for weeks (possibly months). It is pointedout, for the first time, that even though humans (and indeed allvertebrates) mainly excrete their steroids in the form of glucuro-nide and sulfate conjugates, this does not necessarily mean thatthey cannot be accessed by mollusks, as mollusks are one of therichest sources of commercially available glucuronidase and sulfa-tase enzymes.

When one bears in mind that the steroids that everyone hasbeen measuring in mollusks are the same as those made by hu-mans and livestock, one is presented with a far more plausibleexplanation for the presence of steroids in mollusks (i.e. contami-nation). The only observation that still appears be preventing theabandonment of the ‘endogenous origin’ hypothesis is that steroidshave been detected in the flesh of animals that have been sampledfrom what the authors have presumed to be ‘unpolluted rivers’ or‘clean laboratory conditions’. However, the present review pre-sents evidence (some of it new) that everything we, as humans,touch and everywhere we go, we leave traces of ourselves (andthe steroids we have formed). It is concluded that mollusks wouldhave to be reared from eggs in completely sterile conditions withtheir food, water and air being completely devoid of any traces ofvertebrate steroids (whether in free or conjugated form) beforethe presence of steroids in mollusk tissues could be accepted asevidence that they are of endogenous origin.

9. Future directions?

The main thrust of this review has been to point out that thereis no firm proof (i.e. no incontrovertible evidence) that molluskshave an endocrine system based on vertebrate steroids. Some peo-ple will undoubtedly argue that the debate cannot be concludeduntil it can be definitively proved that mollusks do not have theability to make vertebrate steroids. In order to do this, it wouldprobably be necessary to revisit one or two of the species that havealready been reported to carry out critical transformations ofradioactive vertebrate steroid precursors in good yields (in boldtype in Table 2) in order to see whether the results can be indepen-

dently repeated. One referee of the present review suggested theuse of deuterated water (as opposed to 3H or 14C precursors), sothat any labeled steroids (if they were made) could be simulta-neously detected and definitively identified by mass spectrometry.

The fact that the tissues of some molluscs have steroid bindingactivity is still something that needs to be investigated, even if thisbinding turns out to have more to do with the sensing of ‘xenobi-otics’ rather than reception of ‘hormones’. If such binding is medi-ated via non-nuclear mechanisms, knowledge of their identity andfunction would be a useful addition to the ongoing debate on therole of such receptors in the physiology and control of hormone-sensitive cancers in humans.

Further investigations are also obviously required on the abilityof mollusks to absorb, esterify and ‘store’ steroids such as T and E2,even if these investigations cannot prove or disprove that thesesteroids act as hormones in these animals. Apart from helping toreveal the physiological importance of this mechanism for mol-lusks, it is important to know how mollusks deal with the manyother steroids that they are likely to encounter in their environ-ment (such as cortisol, P and EE2). The possibility that humansmight be the source of steroids found in laboratory stocks of mol-lusks is obviously also something that needs to be investigated inmore detail.

Acknowledgements

None of the opinions expressed in this review are intended aspersonal criticisms of any individual or organization. The authorhas experience working with steroids and steroid binding moietiesin fish and cyclostomes, but not directly in mollusks. The authorespecially thanks Dr. Alan Pickering and Dr. Christina Lye for refer-eeing the manuscript (and providing many useful suggestions)prior to its submission. The author also expresses gratitude to Dr.Mike Roberts of the Chemicals and Nanotechnology Division, Defra,UK, for encouraging the project and providing the necessary finan-cial assistance; and to Paul Haywood of Cefas, Weymouth for pro-viding efficient administration.

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