Aciu Zoologicu (Stockh.), Vol. 66. No. 3, pp. 145-150, 1985 Printed in Great Britain

0001-7272185 $3.00+ .OO Pergamon Press Ltd.

0 1985 The Royal Swedish Academy of Sciences

Estimation of Steroids and Steroid Metabolism in a Lower Vertebrate (Lampetra planeri Bloch)

Karl Seiler', Ruth Seiler', Wilfried Ackermann' and Rainer C l a d

'Department of Cell Biology and Regulation, 'Institute of Clinical Chemistry and Laboratory Diagnostic. and 3Department of Biochemistry, Karl-Marx University, Leipzig, G.D.R. (Received 31 January 1985)

Abstract Seiler, K . , Seiler, R . , Ackermann, W. , Claus, R. 1985. Estimation of steroids and steroid metabolism in a lower vertebrate (Lampetra planeri Bloch). (Department of Cell Biology and Regulation, Institute of Clinical Chemistry and Laboratory Diagnostic, and Department of Biochemistry, Karl-Marx University Leipzig, G.D.R.)-Acfa 2001.

(Stockh.) 66, 145-150.

Pregnenolone, androstenedione and testosterone were identified by RIA in tissue homogenates of the pronephric region, the opisthonephros, the gonads and in plasma samples from male and female immature and mature adult brook lampreys. Additionally, hydroxysteroid dehydrogenase activity was determined spectrophotometrically in homogenates from the same tissues of mature and spent adult brook lampreys employing pregnenolone, testosterone or 3p ,17p-dihydroxy-Sa-androstane as suhstrates. The steroid levels show differences corresponding to developmental stages, tissues and sex. Remarkable quantities of testosterone were measured in the testicular tissue homogenates, in homogenates obtained from the pronephric region and in the serum.

Karl Seiler, Sektion Biowissenschaften, Karl-Marx- Universitat, DDR-7010 Leipzig, Talstrasse 33, German Democratic Republic.

Introduction

To comparative endocrinology, the agnatha (Myxiniformes and Petromyzontiformes) are of special interest because of their position in the system of vertebrates, their specific morphology of the steroidogenic tissue, and their special ontogeny with distinct periods. There are numer- ous results of investigations about steroid meta- bolism and steroidogenic cells in lampreys. After the indirect evidence of enzymes specific for steroid hormone synthesis in tissue homogenates (pronephric region, opisthonephros, gonads) taken from Petromyzon marinus and Lampetra planeri (e.g. Weisbart et al. 1978, Callard et al. 1980, Kime and Callard 1982, Seiler et al. 1983), definitive determination of steroid hormone con- centrations by means of radioimmunoassay both in tissue homogenates and sera of these animals was achieved (Piavis et al. 1975, Katz et al. 1982,

Ackermann et al. 1984, Dashow et al. 1984). It was not possible up to now to determine in

which cells these metabolic processes take place, although cells exist in the examined organs whose fine structure is nearly the same as in definite steroidogenic cells (smooth endoplasmic reticu- lum, mitochondria with tubular structures, lipid droplets) (Busson-Mabillot 1967, Hardisty and Baines 1971, Barnes and Hardisty 1972, Youson 1972, Seiler ef al. 1973, Weisbart et al. 1978). These cells are therefore termed presumed ster- oidogenic cells. It is not clear, however, which steroids are produced in the presumed steroid- producing cells, and whether these cells synthe- size different levels of traceable steroid hor- mones in the various organs, at the various developmental stages, in males and females.

In the present study brook lampreys im- mediately before, during and after spawning were of special interest.

145

146 Karl Seiler e t al.

Materials and Methods majority of the assays. The demonstration of pregnenolone and testosterone by RIA points to the fact that these enzyme systems act in the normal metabolism of lampreys, too.

Due to results produced by using different methods it may be concluded with a high degree of probability that presumed steroidogenic cells are indeed the places of synthesis of the demons- trated steroids. This correlation has not been proved with histochemical methods up to now. There are a few identifications of HSD activity in frozen sections of testicular tissue (Hardisty and Barnes 1968, Barnes and Hardisty 1972), ovaries (Hardisty and Barnes 1968, Seiler et al. 1981) and in the presumed adrenocortical tissue of the pronephric region (Seiler et a/. 1981), but numer- ous attempts resulted in insignificant or no de- position of diformazan granules over the pre- sumed steroidogenic cells (e.g. Busson-Mabillot 1967, Seiler et al. 1970, 1973, Youson 1972, Weisbart et a/ . 1978).

In nearly all samples of the same tissue from animals of different developmental stages diffe- rent quantities of the same steroid hormone were measured (Fig. 1). Testosterone decreases in the pronephric tissue of male and female brook lampreys from February to April. In the testis, increasing levels of the same steroid were de- tected. Parkinson (1969, as cited by Barnes and Hardisty 1972) was able to detect testosterone in the testicular extracts obtained from Lampetra Jluviatilis in increasing levels, too. This is in good agreement with the increase in the number of interstitial cells in the testis of LampetraJluviatilis during their development (Barnes and Hardisty 1972). In the ovaries the quantities of testoster- one are constantly rather low over the period of investigation. A clearly demonstrable decrease of testosterone could be shown in three consecutive measurements of opisthonephric tissue of male brook lampreys.

Relatively high values of testosterone were demonstrated by RIA in testicular tissue of ma- ture adult brook lampreys. On the other hand, the enzyme activities measured spectropho- tometrically in the same tissues using testoster- one as substrate are low in lampreys of the same developmental stage. From these spectropho- tometrical measurements the conclusion can be drawn that further metabolism of testosterone takes place probably without dehydrogenases. In this connection it is of interest that spectropho- tometric analysis in testes homogenates of ma- ture adult animals using 3p,l7p-dihydroxy-5a- androstane as substrate leads to a higher value (Table 1). In our previous investigations, (Seiler et a / . 1983) 0.040 nmol min-' mg-' with NAD and 0.048 nmol min-' mg-' with NADP with the same substrate were measured in testes homogenates from mature adult brook lampreys.

Animals

Brook lampreys (Lampetra pluneri Bloch) were caught in October and December 1982 in the little river Plane (Flaming) and killed in February and March 1983 (immature adult), and April 1983 (mature adult) for investigations by radioimmunoassay-mass spectrometry in serum and tissue homogenates. Additionally, anim- als caught in the Plane river in October 1983 were prepared in May 1984 (mature adult) and animals caught during spawning in the Haselbach (Erzgebirge) in June 1984 were prepared some days later (spent animals) for spectrophotometric analysis of hydroxy- steroid dehydrogenase activity in tissue homogenates. The lampreys were maintained in basins with a ground layer of silt and aerated running tap water at approx- imately 10°C up to the time of study.

Radioimmunoassay

Collection of blood and preparation of tissue homogen- ates, chemicals, radioactive steroids, antisera, extrac- tion, chromatography and radioimmunoassay have been previously outlined (Ackermann ei al. 1984).

Spec fro photometric analysis of hydroxysteroid dehydrogenase (HSD) activity

Tissue preparation and optical tests were carried out as described by Seiler et al. (1983), with one variation: the tissues were homogenized by treatment with Alcoa for 10 min and centrifuged at 12.000 g for 10 min. The supernatant was employed for the HSD assay.

Results and Discussion

In the pronephros, the opisthonephros and in the gonads of brook lampreys, characterized by the presence of cells which demonstrate all or some characteristics of steroid-producing cells in their ultrastructure, steroids were detected by RIA (Fig. 1). It is necessary to take into consideration that the proportion of these cells is slight in comparison with the total quantity of the investi- gated tissue [see, for example, Figs. 2(a) and (b)]. The value (ng g-' tissue) is given with refer- ence to this total quantity. In tissue samples of the same regions enzyme activities were detected spectrophotometrically using steroids as subs- trates. The results shown in Table 1 are a con- tinuation of our investigations on larval, meta- morphosing (October) and adult (December, February, April) brook lampreys (Seiler et al. 1983). As in our previous investigations, a higher HSD activity was observed with NADP in the

Tab

le 1

. ob

tain

ed f

rom

adu

lt br

ook

lam

prey

s (L

ampe

tra

plan

eri B

loch

) H

ydro

xyst

eroi

d de

hydr

ogen

ase

activ

ity in

poo

led

hom

ogen

ates

of

the

gona

ds, t

he o

pist

hone

phro

s and

the

pron

ephr

ic r

egio

n

Gon

ads

Opi

stho

neph

ros

Pron

ephr

ic r

egio

n

? d

0 d

0 d

Ster

oid

subs

trat

e C

oenz

yme

Mat

ure

Spen

t M

atur

e Sp

ent

Mat

ure

Spen

t M

atur

e Sp

ent

Mat

ure

Spen

t M

atur

e Sp

ent

Preg

neno

lone

N

AD

n.

d.

n.d.

n.

d.

n.d.

0.

007

n.d.

0.

030

n.d.

n.

d.

n.d.

0.

017

n.d.

N

AD

P n.

d.

n.d.

n.

d.

n.d.

0.

010

n.d.

n.

d.

n.d.

0.

075

n.d.

0.

013

0.01

0

n.d.

n.

d.

Test

oste

rone

N

AD

n.

d.

0.08

5 n.

d.

n.d.

n.

d.

n.d.

n.

d.

n.d.

N

AD

P n.

d.

n.d.

0.

004

n.d.

n.

d.

n.d.

n.

d.

n.d.

n.

d.

n.d.

0.

013

0.01

8

36, 1

76-d

i hyd

roxy

- N

AD

n.

d.

n.d.

n.

d.

0.00

6 0.

017

n.d.

0.

037

0.06

1 n.

d.

0.08

0 n.

d.

n.d.

5a

-and

rost

ane

NA

DP

0.00

4 0.

032

0.01

8 0.

012

0.06

5 n.

d.

0.03

1 0.

030

n.d.

0.

030

0.03

4 0.

070

Not

e-V

alue

s ar

e ex

pres

sed

as n

mol

min

-' m

g-'.

Enzy

me

activ

ity is

bas

ed o

n sp

ectr

opho

tom

etri

cal m

easu

rem

ent (

340 nm)

of N

AD

H

and

NA

DP

H p

rodu

ctio

n, r

espe

ctiv

ely.

Val

ues

repr

esen

t the

mea

ns o

f tr

iplic

ate

dete

rmin

atio

ns. E

ach

assa

y w

as r

un a

gain

st a

bla

nk

whi

ch la

cked

ster

oid

subs

trat

e.

Mat

ure

adul

t: n

= 1

4 d, 14

9.

Spen

t adu

lt: n

= 1

0 d, 3 9.

n.

d. n

ot d

etec

tabl

e.

n.d.

n.

d.

148 Karl Seiler et al.

Tissue homogenates Serum

ng/ml serum

Testosterone

Pregnenolone

Androstenedione

n.d. I

February March

I m m a t u r e adu l t n =ti n = 4

Q

nln

nL

" !

April

ng/g tissue

300

250

February

Ir 1, n, March

Mature adult Immature adult n =9 n = 4 n = 4

a n n.d.

Apri l

5 1

1 oclo

L 2 g g : = Mature adult L r a n - 2 n=IO n = 8 n=ll

Fig. 1 . Concentrations of testosterone, pregnenolone and androstenedione in homogenates of the gonads, the opisthonephros, the pronephric region and in the >era obtained from adult Lamperru pluneri as determined by RIA. Radioactive steroids (New England Nuclear Corp.): [7-'HI-pregnenolone (92.5 X 10"' Bq/ mmol); [ 1,2,6,7-'H]-testosterone (370 X 10"' Bqimmol); [ 1,2-'H]-androstenedione (169.83 x 10"' Bq/mmol). 0 Gonads; opisthonephros; Ppronephric region (only investigated with testosterone); n.d. not detectable. The tissue samples were pooled in each month for the three different regions and for males and females. The blood of male and female animals of each investigation group was pooled because of the very low quantity of obtainable blood (about 50 pJanima1).

These results could show the direction of the further metabolic pathway of testosterone in Larnpetra planeri. Testicular tissue of Larnpetra fiuviatilis is able to convert testosterone (84.4%) in 15P-hydroxytestosterone (Kime and Rafter 1981). Kime and Callard (1982) showed the ability of testicular tissue of sexually mature Petrornyzon rnarinus t o convert 3H-andro- stenedione in 15a-hydroxytestosterone.

In agreement with Ackermann et al. (1984), pregnenolone as a basic component in the meta- bolism of steroid hormones was estimated in all investigated tissue samples. As compared with testosterone, the quantities of pregnenolone and androstenedione detected by RIA in the diffe- rent tissues of brook lampreys show slight varia- tions. The inability to detect androstenedione in the ovaries of all investigated developmental

stages is evident. Kime and Callard (1982) de- monstrated the conversion of l5a-hydroxy- androstenedione from 3H-androstenedione in ovaries of sexually mature Petrornyzon rnarinus. T h e y concluded t h a t a very act ive 15a- hydroxylase system exists in the steroidogenic cells of lampreys. The inability to detect andros- tenedione in ovaries of brook lampreys by RIA could be caused by this fact.

These examples show some possibilities of steroidogenesis in lampreys.

The quantities of steroids measured in the serum (Fig. 1) confirm the findings in the tissue homogenates. The high values of traceable tes- tosterone in March and April are evident. They are in agreement with our previous investigations (Ackermann et al. 1984) on adult Larnpetra planeri (March; 20 ng/ml serum).

Steroids in Brook Lampreys 149

Fig. 2. Lumpetru pluneri. Presumed steroid-producing tissues. (a) Pronephric region-longitudinal section. Arrows presumed adrenocortical tissue. (b) Opis- thonephros--cross section. Arrows presumed adrenocortical tissue. Mature adult. Fixation with Bouin's solution, wax embedding with tissuemat, section of 5 pm thickness, trichrome staining (Goldner). x 200.

150 Karl Seiler et a1

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