effects of estrone and estradiol-17β on 25-hydroxycholecalciferol hydroxylase activities in female...

GENERAL AND COMPARATIVE ENDOCRINOLOGY 71, 97-104 (1988)

Effects of Estrone and Estradiol-17p on 25Hydroxycholecalciferol Hydroxylase Activities in Female Japanese Quail

P. J. WILLIAMS,' P. C. BRAIN, M. J. PEDDIE,~ AND T. G. TAYLOR~

School of Biochemical and Physiological Sciences, University of Southampton, Southampton SO9 3TU, England

Accepted January 29, 1988

Equimolar amounts of estrone and estradiol-17B injected into sexually immature female Japanese quail caused comparable increases and decreases in the renal activity of 25 hydroxycholecalciferol (25HCC) la-hydroxylase and 2Shydroxycholecalciferol 24- hydroxylase, respectively. Peak activity of the former enzyme was induced by both estrogens within 6 hr and had declined by 24 hr. Plasma concentrations of estrone and estradiol following the injection were maximal within 3-6 hr of injecting the steroid i.m. Following [3H]estradiol injection, 25% of the radioactivity was located with estrone in the plasma following separation by TLC. Conversely, following [3H]estrone injection 30% of the radioactivity in plasma was located with estradiol. Thus, both estrone and estradiol may have physiological effects in quail, which in part may be due to their in vivo interconversion. Evidence that prolactin may be mediating the estrogenic stimulation of the 25HCC la-hydroxylase is also presented. o 1988 Academic PWX~, hc.

It has been recognized in recent years that 1,25dihydroxycholecalciferol is the major active metabolite of vitamin D in avian and mammalian species and that a key enzyme in controlling its synthesis, 25 hydroxychlolecalciferol la-hydroxylase (25HCC la-hydroxylase) is regulated by numerous factors including, in birds, estrogens (Baksi and Kenny, 1978; Sedrani et al., 1981). The 25HCC may also be metab- olised to other physiologically less active hydroxylated products; in quail the pre- dominant hydroxylase is 25-hydroxylchole- calciferol 24-hydroxylase (25HCC 24- hydroxylase). The activity of these two enzymes is often reciprocal (Sedrani et al., 1981). The activity of the renal 25HCC la-hydroxylase enzyme closely parallels the changes in level of 1,25-dihydroxycholecalciferol in the plasma (Spanos et al.,

’ Current address: Department of Biochemistry, University of Oxford.

’ To whom correspondence should be addressed. 3 Deceased.

1976b) and is often used as an index of control for this reason. 1,25-DHCC in turn causes hypercalcaemia by its actions on in- testinal calcium transport, bone resorption, and renal tubular reabsorption (De Luca, 1979).

In birds, estrogen-stimulated renal la-hydroxylase activity is particularly im- portant; in quail, the activity of these enzyme rises rapidly during sexual maturation (Turner et al., 1979). Furthermore, there is an increase in plasma estradiol and estrone at approximately the same time (Brain et ul., 1988). It was of interest, therefore, to see whether estrone as well as estradiol could stimulate renal 25HCC lol-hydroxylase activity in vivo, in birds in which this enzyme is otherwise relatively inactive, and to compare the enzyme activity with changes in plasma calcium levels and estrogen concentrations. In addition, two estrogens not normally found in quail plasma, estradiol-17a and estriol, were used in some experiments.

The mechanism by which estradiol regu-

97 0016~6480/88 $1.50 Copyright 0 1988 by Academic Press, Inc. All rights of reproduction in any form reserved.

98 WILLIAMS ET AL.

lates renal 25HCC hydroxylase activity has not been elucidated; it does not have any direct action in vitro (Spanos et al., 1978). The initial experiments in this work revealed a very rapid time course for the effects of the estrogens, within 3-6 hr, which raised the possibility that estrogens might act indirectly. Since it is known that estrogens stimulate pituitary prolactin release in birds (McNeilly et al., 1978) and that prolactin stimulates renal 25HCC lol-hydroxylase activity also (Spanos et al., 1976a, Baksi et al., 1978), the effect of a known antagonist of pituitary prolactin secretion, bromocryptine, upon the response to estradiol was investigated.

METHODS

Birds and Treatment Regimes

Japanese quail, Xl-70 days old, raised on 8 hr light/24 hr, were used in this study, from the colony described previously (Brain et al., 1988).

Experiment 1. Six or seven female birds were injected i.m. with 1.8 umol/kg estrone, estradiol-17B estradiol-17cY, or estriol in ethyl oleate (approximately 100 pJ/injection) for 3 consecutive days; blood samples were withdrawn from the wing vein immediately before each injection; the birds were killed 24 hr after the last injection by decapitation; ovaries and oviducts were removed at this time and weighed; the kidney tissue was removed for the estimation of 25HCC la-and 24-hydroxylase activities; and plasma calcium, phosphate, estradiol-178, and estrone levels were measured subsequently.

Experiment 2. Female Japanese quail, 50-70 days old, raised on 8 hr light/day, were injected with 1.8 pmoUkg estradiol-17B in ethyl oleate or vehicle alone (n = 6) once daily for 3 days, as in the previous experiments, and killed on the fourth day: blood was collected without exposure to the atmosphere, by car- diac puncture, and ionized calcium levels were determined, as well as total calcium concentration.

Experiment 3. Female quail, raised on 8 hr light/day, when SO-70 days old, were injected i.m. with 1.8 umol/kg body wt of estrone or estradiol-17B in ethyl oleate, or with vehicle alone, in the second hour of their light period. Birds were killed by decapitation 3, 6, 12, or 24 hr after the injection, and blood and kidney tissue collected for further analysis of estrogens and 25HCC hydroxylase activities, respectively.

Experiment 4. Female quail, as above, were injected i.m. with approximately 10 uCi [l,2,6,7-31estrone or

[I ,2,6,7-3H]estradiol-17B in ethanol, which also con- tained nonradioactive estrogen to give a final concentration of 1.8 pmol/kg body wt/injection. The birds were killed 30 min after the injection, trunk blood was collected and centrifuged, and the estrogens were extracted and estrone and estradiol separated as described below.

Experiment 5. Four male and four female quail, raised on 8 hr light/day, were injected i.p. with 500 pg of the dopamine and antagonist 2-bromoergocryptine methane sulphonate/lOO g body wt in 100 ~1 of di- methylformamide:water (1:9, v/v) as vehicle, or with vehicle alone. Thirty minutes later 1.8 pmol/kg body wt of estradiol-17B was injected i.m. in ethyl oleate into the same birds, or into a corresponding group of control birds; a further control group was injected with ethyl oleate and the ergot alkaloid. Five and one-half hours after the estrogen injection the birds were killed and renal tissue was removed for renal 25HCC hydroxylase activity determination.

Blood Analyses

Total calcium levels were measured as described previously (Singh et al., 1986) in 50 pJ plasma using a Perkin-Elmer 280 series atomic absorption flame spec- trophotometer at 423 nm.

Ionized calcium levels were measured in l-2 ml plasma using the calcium-specific electrode on a radi- ometer pH meter, as described previously (Singh ef al., 1986).

Acid labile phosphate was measured in 50 pJ plasma following acidification with trichloracetic acid and buff- ered in ammonium molybdate/perchloric acid/ascorbic acid solution against phosphate standards in a Perkin- Elmer spectrophotemeter at 700 nm.

Estradiol-17P and estrone were measured by radioimmunoassay as described previously (Brain et a/., 1987).

Renal Hydroxylase Assay

Kidneys were removed into ice-cold buffer (0.2 M KH,PO,; 4 mM MgCl,,6H,O; 7.5 mM L-malic acid; 3.5 m&f D-glucose 6-phosphate), blotted, and weighed, and a 10% homogenate (w/v) was prepared, using live passes of a Telfon pestle at 300 rpm. The incubation was started by adding 100 ul homogenate to 1.9 ml cold assay buffer (supplemented with 0.3 mM NADP and 600 ul glucose-6-phosphate dehydrogenase and 6.5 pmol of 25-[3H]hydroxycholecalciferol (the substrate). Incubation were carried out at 37” in a shaking water bath (80 cpm) for 10 min, then stopped by the addition of 8 ml methanol:chloroform (2: 1, v/v), shak- en, stood in the dark for 20 min at room temperature, and centrifuged at 2000 rpm. The pellet was extracted four times with methylene chloride and evaporated un-

ESTROGENS AND VITAMIN D METABOLISM 99

der reduced pressure, and the metabolites were separated by TLC in heptane:ethyl acetate:ethanol, 12: 12: 1 (v/v/v), in the dark at 4”. Nonradioactive standards (25HCC; 24,25-DHCC; and 1,25-DHCC) were also applied to the plate routinely to help locate the radioac- tive metabolites. Other metabolites of 25HCC (1,24,25THCC; 25,25-DHCC) were used initially to verify the separation procedure. The 1 ,25-DHCC and 24,25-DHCC had Rf values of 0.25 and 0.39 in this system. The separate bands were scraped off the TLC plates into scintillation vials and radioactivity was counted and corrected to dpm, and adjustment was made for experimental loss. Overall recovery was approximately 94%.

Further validation of the TLC system was carried out using HPLC, using a single straight phase u-Porasil column with a solvent system of heptane 20:isopropanol 1 and a flow rate of 2 ml/min (800 psi); this revealed that the I,25-DHCC and 24,25-DHCC metabolites separated by TLC were essentially free from contamination by other products.

The substrate concentration and time course of the reactions were studied, and the times employed here were on the linear component of the response curve in each case.

Separation of Estrogens

Plasma was denatured after centrifugation with 3 vol ethanol; a small amount of [i4C]estrogens was added (1000-5000 dpm). Ethanol extracts were applied to TLC plates together with unlabelled estrogens and separated in benzene:ethyl acetate (3: 1, v/v) in which the Rf values for estrone and estradiol were 0.65 and 0.40, respectively. The powder was scraped into scintillation vials and radioactivity counted using a dual label program correcting to disintegrations per minute. The results were expressed as percentage of the total dpm as estradiol and estrone within each peak and were corrected for experimental loss. Recoveries were 60-70%.

Chemicals and Reagents

Analar-grade chemicals were used to prepare standards and buffers for the calcium and phosphate as- says. Other chemicals were reagent grade; they were obtained from BDH Ltd (UK). Vitamin D standards were kindly donated by Hoffman LaRoche (Switzer- land); radiochemicals were obtained from Amersham International (UK); steroids, NADP, D-glucose 6- phosphate, and malic acid were from Sigma Chemical Co. (UK).

RESULTS

Experiment 1

Body and ovarian weights remained un-

changed following estrogen treatment; however, all four estrogens caused an increase in oviduct weight over the experimental period (Table 1). The weight of the oviducts was increased 3-to 4-fold by estradiol-17a, lo-fold by estradiol-17@, and 7-to &fold by estrone treatment.

Total plasma calcium levels were unaf- fected by the experiment in the vehicle treated birds and did not alter after treatment with estradiol-17o or estriol. Howev- er, the levels rose significantly by the third day in the estradiol-17P-treated birds and by the fourth day in the estrone-treated birds (Table 1). By the fourth day, plasma phosphorus levels were also raised in these two groups, but not in the other groups. The renal 25HCC lo-hydroxylase activity was also greater than control values in the estrone and estradiol-17B-treated birds, but not in the other groups. 25HCC 24- hydroxylase activity was not consistently affected by any of the treatments in these birds. Estradiol levels were slightly raised on the fourth day compared with controls in the estradiol-17B-treated group (1.05 IL 0.14 pmol/ml compared with 0.60 + 0.05 pmol/ml in the controls) but not affected in the other groups. Estrone levels were not different between the groups at any time.

Experiment 2

Since the increase in plasma calcium levels in the previous experiment could have been due to increase in either total or ionized calcium levels, the ionized values in birds treated with estradiol-17B for 3 consecutive days were determined. The total concentration was found to be increased in the estradiol-17B-treated birds (4.48 +_ 0.31 mmol/liter) compared with vehicle-treated controls (2.28 k 0.05 mmol/liter, P < 0.001); in contrast, ionized calcium levels were similar in the two groups (1.15 + 0.06 compared with 1.02 f 0.03 mmol/liter, respectively).

TABL

E 1

EFFE

CT

OF E

STRA

DIOL

-170

1, E

STRA

DIOL

-17B

, ES

TRON

E, O

R ES

TRIO

L IN

JECT

ED i.

m.

INTO

QUA

IL

(1.8

pm

ol/k

g BO

DY W

T) O

N TO

TAL

PLAS

MA

CALC

IUM

AND

ACID

LA

BILE

PHOS

PHAT

E LE

VELS

,~TE

RINE

~EIG

HT,A

NDON

TH

E AC

TIVI

TIES

OF

RENA

L%HY

DROX

YCHO

LECA

LCIF

EROL

la-

AN

D %

HYDR

OXYL

ASE

ACTI

VITI

ES

-- ._

____

_ ._

Day

4 -

..__-

To

tal

Day

1 Da

y 2

Day

3 pl

asm

a Pl

asm

a Ov

iduc

t I-H

ydro

xyla

se

24-H

ydro

xyla

se

----

calci

um

phos

phor

us

weig

ht

activ

ity

activ

ity

Q Gr

oup

Tota

l pl

asm

a ca

lcium

m

mol

/liter

(m

mol

/liter

) (m

mol

/liter

) (m

g)

(Pm

oVhr

k)

(pm

ol/hr

/g)

E __

___-

__

_.-

__I

Cont

rol

2.85

i

0.5

2.90

-t

0.08

2.

83 +

0.1

3 2.

80 r

t 0.

13

0.58

ct 0

.03

17.3

-+

1.2

12.1

3 -+

2.

2 14

5.1

I 33

.0

j: is (5

) (5

) Es

tradio

l-17u

2.

75 +

0.2

0 2.

98 r

t 0.

15

3.23

2 0

.10

3.23

rt

0.15

0.

77 i

r 0.

05

64.9

3.r

10.9

**

23.5

0 f

10.3

93

.6 z

t 15.

3 E:

Es

tradio

l-17B

2.

90 2

0.1

5 3.

78 k

0.1

8 6.

35 k

1.

05

10.0

8 rt

1.93

1.

58 +

0.2

2 17

8.6

‘- 40

.2**

36

.90

2 7.

7*

246.

2 rt

90.3

(3

F

Estro

ne

2.90

k 0

.18

3.35

2 0

.25

4.35

+ 0

.45

6.73

rt

0.90

1.

13 +

0.1

7 13

5.8

z!z 1

0.3*

* 25

.70

k 4.

4*

162.

0 2

24.5

(7

) (7

) Es

triol

2.

75 2

0.0

8 3.

00 z

!c 0.

05

3.18

ir

0.25

3.

10 r

t 0.

10

0.63

t:

0.09

42

.5 l

r 9.

2*

24.9

0 2

8.4

144.

1 ir

29.2

__

__

Note

. Es

troge

ns

were

inje

cted

daily

fo

r 3

days

i.m

. int

o fe

mal

e Ja

pane

se

quail

ra

ised

on 8

hr

light

/day

. Bl

ood

was

colle

cted

fro

m

a wi

ng

vein

imm

edia

tely

prior

to

the

inj

ectio

n.

Trun

k blo

od

was

colle

cted

fo

llowi

ng

deca

pitat

ion

on D

ay 4

and

kid

neys

we

re

rem

oved

fo

r as

say

of h

ydro

xyla

se

activ

ities

, as

des

cribe

d in

the

text

. To

tal

calci

um

leve

ls an

d ac

id lab

ile

phos

phat

e le

vels

were

mea

sure

d sp

ectro

phot

omet

rical

ly as

des

cribe

d in

the

text

. Re

sults

are

exp

ress

ed

as M

+ S

EM w

ith

n =

6 in

each

gro

up

unle

ss o

ther

wise

st

ated

(in

pare

nthe

ses)

. Si

gnific

ant

diffe

renc

es

of th

e tre

ated

bir

ds f

rom

th

e co

ntro

l bir

ds a

re s

hown

(*P

< 0

.05;

**P

< 0

.01)

as

dete

rmine

d by

the

Stu

dent

’s t t

est.


Experiment 3

The activity of the renal 25HCC la-hydroxylase increased significantly in both estrone and estradiol-17@injected birds by the third hour postinjection and continued to rise to a maximum 6 hr postinjection; the activity of the 25HCC la-hydroxylase had returned to values slightly greater than control by 24 hr postinjection (Table 2). In contrast, 25HCC 24- hydroxylase activity decreased significantly in the estrogen-treated birds to a nadir at 12 hr; by 24 hr, the 25HCC 24-hydroxylase activity was similar to the pretreatment value.

Plasma estrone levels in the estrone- treated birds rose to a peak by 6 hr after the injection, falling again by 12 hr; plasma estradiol levels also increased slightly in this group following the estrone injection. Fol- lowing the injection of estradiol-17l3, estradiol levels in plasma peaked 3 hr after the injection and plasma estrone levels were raised at the same time although to a lesser extent (Table 2).

Experiment 4

There was evidence for substantial inter-

conversion of estradiol and estrone in the plasma of the quail, following the injection of the radiolabelled steroid. Following injection of [3H]estradiol-1713, 23.8 + 2.8% (n = 5) of the total estradiol + estrone recovered from the plasma was found as estrone. Conversely, following 13H]estrone injection, 30.5 ? 2.9% (n = 5) of the estrone + estradiol recovered from plasma was located with estradiol.

These are preliminary, nonsteady-state experiments, but do confirm some of the suppositions raised by previous experiments.

Experiment 5

The data for male and female birds have been combined since they were not different from each other; estradiol-17P injection resulted in an increased level of 25HCC la-hydroxylase activity and reduced 25HCC 24-hydroxylase activity, as in previous experiments; however, both effects of the estrogen were almost completely abolished by the bromocryptine injected before the estrogen. Neither bromocyrptine alone nor the two vehicles had any appre-

TABLE2 RENAL~~-HYDROXYCHOLECALCIFEROL ~WHYDROXYLASEAND 2bHYDROXYLASE ACTIVITY AND PLASMA

ESTRADIOL AND ESTRONE CONCENTRATIONS ON QUAIL INJECTED WITH 1.8 p,mol/kg ESTRADIOL OR ESTRONE

Injection

Time after injection

(hr) Number of

observations

Renal hydroxylase activity (pmoUhr/g)

I-Hydroxylase 2bHydroxylase Estradiol

Estradiol-l7p 0 3 6

12 24

Es&one 0 3 6

12 24

Ethyl oleate 6 Nothing 6

9 6.3 k 1.9 159.4 + 32.7 31.7 k 7.5** 69.5 T 9.4**

178.0 2 34.3*** 87.3 t 24.8 129.6 k 24.3*** 35.1 t 5.9*** 45.9 k 7.8’* 246.3 + 89.7

6.3 + 1.9 159.4 ? 3.7 100.9 2 1.0*** 138.1 k 66.1 172.9 + 36.9*** 53.8 + 16.8* 62.8 -c 10.8** 28.5 + 4.6** 80.9 k 23.4** 214.8 k 61.3

9.4 t 6.4 210.7 + 47.6 3.1 + 1.7 233.8 + 83.9

Plasma steroid

1.54 + 0.29 12.45 + 1.86***

7.99 + 2.44* 3.67 f 0.26*** 1.94 + 0.29 1.54 + 0.29

4.06 i 0.73* 3.54 ‘- 0.43** 1.30 2 0.46 1.79 + 0.23

- -

3.98 2 0.58 12.54 f 0.86*** 12.48 t 2.70*

3.34 ‘- 0.54 3.01 -t 0.49 3.98 2 0.58

19.80 k 3.08** 16.13 It: 1.63** 4.75 2 1.87 4.14 t 0.45

- -

Note. Female quail aged 50-70 days, raised on 8 hr light/day, were injected i.m. with 1.8 PmoVkg body wt estradiol-17s or estrone, with vehicle or not at all. They were killed by decapitation at intervals after the injection as indicated and trunk blood and kidneys taken for analysis of estrogens or hydroxylase activities as described in the text. Results are expressed as M ? SEM; differences from the preinjection control value are indicated (*P < 0.05, **P < 0.01; ***I’ < 0.001) as evaluated by the Student’s t test.

102 WILLIAMS ET AL.

ciable effect on the hydroxylase activities (Table 3).

DISCUSSION

The present results show that both estrone and estradiol can have potent effects on renal 25hydroxycholecalciferol hydroxylases in Japanese quail. Estrone, like estradiol-17B, also stimulates an increase in oviduct weight, whereas other estrogens (estradiol-17a and estriol) are less effective in this respect. Furthermore, both estrone and estradiol can cause a rise in plasma calcium levels, although this must be largely attributed to the increase in the plasma- bound calcium component (nonionized component); it is known that estradiol injected into roosters causes an increase in hepatic protein synthesis (largely egg yolk protein) (Hertelendy and Taylor, 196.5), which has a high calcium-binding capacity.

The time course of the action of estrone and estradiol upon the renal 25HCC hydroxylases is relatively rapid and closely parallels the rise in plasma steroid levels which follows the injection of the free steroid. Differences between the present and previous work (Baksi and Kenny, 1978; Se-

TABLE 3 RENAL 25HCC HYDROXYLASE ACTIVITIES IN QUAIL

INJECTED WITH BROMOERGOCRYPTINE AND/OR ESTRADIOL-17P

- Hydroxylase activities

Treatment I-Hydroxylase 24-Hydroxylase

(pmofirk) (pmofirk)

Vehicle 1 + vehicle 2 8.4 2 3.5 260.4 +- 32.4 Ergot + vehicle 2 11.8 f 3.4 294.3 2 57.6 Ergot + estradiol-17e 53.5 k 10.6 175.1 + 53.2’ Vehicle 1 + estradioLl7e 210.5 + 46.8** 51.7 -t 11.5**

Note. Eight quail, raised on 8 hr light/24 hr, were injected i.p. in the second hour of their light period with 500 pg 2- bromoergocryptine methyl sulphonatellO0 g body wt in 100 ~1 of dimethylformamidewater (1:9, v/v) (vehicle 1). Thirty minutes later they were injected with 1.8 kmolikg body wt estradioL17P in ethyl oleate (vehicle 2). Controls were injected with vehicle 1 and/or 2, as indicated. They were killed 5% hr after the estradiol injection and kidney tissue was taken for the determination of 25HCC hydroxylase activities as described in the text. Results are expressed as M + SEM (n = 8); *P -C 0.05 and **P < 0.01 indicate significant levels based on com- parison of data with the vehicle 1 and vehicle 2 treated birds by the Student’s t test.

drani et al., 198 1) may be explained to some extent by our use of the free steroid while others have used a conjugated form. Pre- liminary experiments showed that estradiol injections had not caused a rise in 25HCC la-hydroxylase activity within 2 hr in sexually immature quail. It might be of interest to measure the changes in ionized calcium levels which should occur with the changes in lo-hydroxylase activity, although evidently the homeostatic mechanisms in the bird are capable of maintaining plasma ionized calcium levels over a 24-hr period. There are quite marked fluctuations in plasma ionized calcium levels during the ovulatory cycle in hens (Singh et al., 1986), but similar information on physiological variations in quail is not available. The compensatory mechanisms employed by the quail presumably include calcitonin, which has not been measured, and reten- tion of phosphate by the renal tubules, since plasma phosphate was elevated by estrogens in these experiments. Thus the response to the acute injection of estrogens, once or once every 24 hr, is somewhat ar- tificial; the levels of estrogen attained postinjection were substantially greater, for a short period of time, than those found in sexually mature birds (Brain et al., 1988), but the steroid was evidently cleared rapidly from the blood. Repeated daily injections of estradiol-17P had a cumulative effect on plasma calcium and phosphate levels; whether these were similar to or greater than the phosphate and total calcium levels found in sexually mature female quail we do not know at this time. The dose of estradiol-17B used here was known to be sufficient to stimulate 1 a-hydroxylase at the 6-hr time; the dose was equivalent to 490 l&kg for estradiol. In preliminary experiments we found that 31 Kg/kg had very little effect on the 25HCC la-hydroxylase; 122 kg had a half maximal effect, while 1961 &kg had only a slightly greater effect than the 490 pglkg dose.

It is evident that the 25HCC la-hydroxylase and 25HCC 24-hydroxylase respond


in a different time course and in opposite ways to the injected estrogen; the suppres- sion of 24-hydroxylase may be a direct response to the estrogens, whereas the sub- sequent rise may be due to elevated, 1,25- DHCC levels which is a most potent stimulator of the 24-hydroxylase (Henry, 1979). Similar reciprocity in the control of these enzymes has been noted previously (Sedrani er al., 1981).

There is evidently substantial interconversion of the two estrogens which were found to affect the renal hydroxylases and stimulate uterine weight. The present evidence is insufficient per se to say that only estrone converted to estradiol is capable of a physiological action on the target tissues; nevertheless, it does emphasize that estrone in the plasma may well have a physiological role in quail, especially since the estrone concentration in mature birds is very similar to that of estradiol (Gulati et al., 1980; Doi et al., 1981; Brain et al., 1987).

During sexual maturation of female quail, both estrone and estradiol rise in the plasma (Brain et al., 1987), and there is a rise in renal 25HCC lo-hydroxylase activity (Turner et al., 1979). The two events may be causally related, but renal 25HCC hydroxylase activity in ovariectomized female birds has not yet been studied.

It is noteworthy that the time course of the effect of the estradiol is very rapid, rather more so than might be anticipated for the classic model of the action of a steroid hormone. This suggested that estradiol might be acting through an alternative, pos- sibly indirect, route, and the possibility that prolactin might be implicated was investigated. It is known that estradiol stimulates prolactin secretion in turkeys (McNeilly et al., 1978), and dopamine may have an in- hibitory role in regulating prolactin secretion in birds (Harvey et al., 1982). More- over, prolactin is capable of stimulating renal 25HCC la-hydroxylase activity in avian kidneys boths in vivo and in vitro (Spanos et al., 1976a; Bikle et al., 1980; Spanos et al.,

1981). Furthermore, a direct action of estradiol on renal 25HCC hydroxylase activity has not been demonstrated (Spanos et al., 1978). Lastly, it may not be uncon- nected that both plasma estrogens (Brain et al., 1988) and pituitary prolactin content (Goldsmith and Hall, 1980) rise during sexual maturation in female quail, with a similar time course to the increase in renal 25HCC la-hydroxylase activity (Turner et al., 1979; Sedrani et al., 1981). In experiment 5 the effect of estradiol-17P injection was reduced by the prior injection of bromoergocryptine. This compound is known to reduce prolactin secretion in mammals (D’Agata and Scapagnini, 1979), although a directly comparable effect in birds has not been demonstrated. We were not able to measure prolactin directly in these experiments, but to do so would be valuable.

Thus, in conclusion, we can say that estrone as well as estradiol may have a role in regulating renal 25HCC hydroxylase activities, there is some evidence for interconversion of these two steroids in viva; the mechanism of action of the estrogens may be indirect on the renal 25HCC hydroxylases and prolactin is implicated.

ACKNOWLEDGMENTS

This research was funded in part by grants from the ARFC and BEMB.

REFERENCES

Baksi, S. M., and Kenny, A. D. (1978). Vitamin D metabolism in Japanese quail: Gonadal hormones and dietary calcium effects. Amer. J. Physiol. 234, E622-E628.

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effects of estrone and estradiol-17β on 25-hydroxycholecalciferol hydroxylase activities in female...

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