Comp. Biochem. Physiol. Vol. 90B, No. 1, pp. 221-225, 1988 0305-0491/88 $3.00 + 0.00 Printed in Great Britain © 1988 Pergamon Press pie

STEROID REGULATION OF KIDNEY HISTIDINE DECARBOXYLASE AND ORNITHINE DECARBOXYLASE

LEVELS IN MOUSE KIDNEY: EFFECTS OF THE MUTATION TESTICULAR FEMINIZATION, Tfm

RICHARD J. MIDDLETON* and GRAHAME BULFIELD t Gene Expression Group, AFRC Institute of Animal Physiology and Genetics,

Edinburgh Research Station, Roslin, EH25 9PS, Midlothian, Scotland, UK (Tel.: 031-440-2726)

(Received 5 June 1987)

Abstract--1. Testosterone represses kidney histidine decarboxylase levels in both normal male and female mice. Tfm/Y mutant mice lack an androgen receptor and are phenotypically female. It has been suggested that the testosterone induction of HDC levels in these animals is a result of aromatisation to oestrogens in the absence of the androgen receptor; the oestrogens then induce the enzyme.

2. It is shown that the induction of HDC in Tfm/Y mice is specific to testosterone and not other androgens and can be mimiced by low doses of fl-oestradiol in normal female mice.

3. Analysis of Tfm/+ mice indicates that the testosterone induction effect is a function of individual kidney cells.

INTRODUCTION

The interactions between hormones and genes in mammals are not yet fully understood. Mutations, such as the murine X-chromosome linked gene Testi- cular Feminization (Tfm), which affect hormone sen- sitivity, therefore, provide important tools to analyse hormonal regulation of gene expression. Tfm causes a general androgen insensitivity (Lyon and Hawkes, 1970; Ohno and Lyon, 1970); TfmfY males have small testes, but they develop completely female secondary sexual characteristics. The primary lesion caused by the mutation is a deficiency of the cystolic androgen-receptor complex (Gehring et al., 1971; Bullock and Bardin, 1974; Attardi and Ohno, 1974; Gehring and Tomkins, 1974) and this results in several kidney androgen sensitive enzymes no longer being modulated by testosterone (Dofuku et al., 1971; Bulfield and Nahum, 1978).

Both ornithine decarboxylase (ODC; EC 4.1.1.1.17) and histidine decarboxylase (HDC; EC 4.1.1.2.2) levels are modulated by androgens in mouse kidney (Kahlson and Rosengren, 1968; Pajunen et al., 1980; Martin et al., 1984). ODC is androgen inducible with normal males having 40-fold the levels of females (Middleton et al., 1987); Tfm/Y animals have female-like levels. HDC levels are, on the other hand, repressed by androgen and induced by oestrogen; producing a sex difference of about 20-fold in favour of females (Bulfield and Nahum, 1978; Martin et al., 1984); Tfm/Y animals, however, instead of having high female-like levels of HDC,

*Present address: Department of Biochemistry, University of Birmingham, Birmingham, B15 2TT, UK.

tAll communications to Dr G. Bulfield.

have low male-like levels (Bulfield and Nahum, 1978). This is so far the only male-like enzyme phenotype discovered in TJ~n/Y animals. Furthermore, instead of HDC levels in TfmfY animals being unaffected by androgen injections, as would be expected, they are induced several hundred fold; this contrasts dramatically with the androgen repression of HDC observed in normal males and females (Bulfield and Nahum, 1978). It was suggested that in the absence of the androgen binding complex in Tfm/Y animals, testosterone might be aromatised to oestrogens which then induced HDC. The induction of kidney HDC by fl-oestradiol in normal female mice is, however, only of the order of two- to three-fold (Martin et al., 1984), suggesting that a different mechanism may be operating to cause the massive induction in Tfm/Y animals.

We have investigated the modulation of HDC and ODC levels by a variety of androgens in Tfm/Y mice and made attempts to reproduce the "super-induction" effect of testosterone (seen in Tfm/Y animals) in normal females by various hor- mone manipulations and to identify the mechanisms involved.

MATERIALS AND METHODS

C57BL/10 mice were obtained from Bantin and Kingrnan Ltd, Grimston, Hull, UK. The Tfm stock is kept in this laboratory and the gene has been made congenic on a C57BL/10 background by repeated backcrossing (over 20 times). All chemicals were obtained from Fisons or Sigma except where stated. All hormone treatments were by subcu- taneous implantation of pellets (with the exception of fl-oestradiol in which two further methods were employed). The pellets were made by compacting 30 mg of the hormone with 5 mg of cellulose, these were then inserted into the

221

222 RICHARD J. MIDDLETON and GRAHAME BULFIELD

Table 1. The effect of steroid implants on histidine deearbosylase and ornithine decarboxylase activities in normal female and testicular feminized male mice

HDC levels ODC levels

Steroid Normal females (N) Tfm/Y mice (N) Normal females (N) Tfm/Y mice (N)

Control 0.30 + 0.08 (3) 0.005 + 0.003 (3) 0.85 + 0.11 (3) 0.95 + 0.21 (3) Testosterone 0.047 + 0.009 (3) 5.97 ___ 1.27 (7) 89.4 + 4.15 (3) 1.03 + 0. t 3 (3) Androstene-4-ene-3,17-dione 0.031 + 0.012 (3) 0.002 + 0.001 (3) 108 + 9.52 (3) 1.06 + 0.15 (3) 5~t-Dihydrotestosterone 0.009 _+ 0.003 (3) 0.009 _+ 0.005 (3) 94.3 -+_ 3.63 (3) 0.83 + 0.27 (3) 5ct-Androstane-3,17-dione 0.041 + 0.025 (3) 0.025 + 0.010 (3) 41.6 _+ 8.56 (3) 0.94 +_ 0.28 (3) /~-Oestradiol 0.60 _+ 0.20 (3) 0.35 + 0.02 (3) 0.63 + 0.21 (3) 0.80 + 0.11 (3) Thyroxine 20.4 + 1.32 (3) 19.1 + 2.49 (3) 0.89 ± 0.12 (3) 1.14 + 0.07 (3)

animal's neck by making a small nick and were pushed down the slit under the skin to lie in the animal's lower back; thyroxine was implanted for ten days, the androgens for seven and the oestrogens for fourteen days.

fl-Oestradiol was also administered by means of poly- ethylene capsules. Two sizes were made, 0.5 cm in length (referred to as "small") and 1.0 cm (large) using Intramedic PE-200 tubing (i.d. 1.40 mm; o.d. 1.90 mm; Dow Coming, Midland, MI, USA); the procedure for filling the capsules was basically that described by Butterstein et al. (1980). The capsules were implanted subcutaneously and left for seven days. In addition to the two types of implants, 13-oestradiol was dispersed in olive oil and 100 #1 was injected intra- peritonally, daily between 9.00 am-10.00 am for periods of up to fourteen days (see text). Animals injected with olive oil alone, showed no detectable alteration in HDC levels compared with control animals (results not shown).

HDC and ODC activities were determined by the release of '4CO2 from L-carboxyl 14C histidine or ornithine (Am- ersham International, UK); and are expressed as nmol of substrates utilised/min per g wet wt of kidney at 30°C; details of the assay conditions have been previously de- scribed (Martin et al., 1984; Middleton et al., 1987).

RESULTS

Effects of the mutation Tfm on the response of kidney HDC and ODC to steroid hormones

Histidine decarboxylase levels in normal female mice are repressed by implants of testosterone. Other androgens also repress H D C activity, with 5~-dihy- drotestosterone being the most potent (Table 1). ]~-Oestradiol implants increase H D C levels by two- fold but the most dramatic induction of H D C is by thyroxine where over 60-fold induction is observed (see also Mart in et al., 1984).

In Tfm/Y individuals, which normally have male- like levels of HDC, a similar pattern of induction by /~-oestradiol and thyroxine is observed. When implanted with androgens, however, Tfm/Y animals behave very differently; H D C is induced over 1000- fold by testosterone (5.97 units, cf. 0.005; Table 1)

although implants of three other androgens 5ct- androstane-3,17-dione, androstene-4-ene-3,17-dione and 5~-dihydrotestosterone do not have a significant effect.

In normal females, ornithine decarboxylase levels are dramatically induced by implants of testoster- one (Table 1). Of the three other androgens tested, both androstene-4-ene-3,17-dione and 5ct-dihydro- testosterone also induced O D C levels over 100-fold, al though 5~t-androstane-3,17-dione induced O D C rather less (49-fold); fl-oestradiol and thyroxine had little or no effect on O D C levels in normal female mice (Table 1). O D C in Tfm/Y animals shows female-like levels; none of the hormone treatments used had any effect (Table 1).

Induction of HDC by testosterone in female littermates of Tfm/Y animals

The induction of H D C by testosterone Tfm/Y mice was further investigated in female littermates of Tfm/Y animals. The H D C levels in these + / ? animals after testosterone implantation are higher than those of + / + animals although with a large standard error (Table 2, t = 3.77 P < 0.01); the large standard error is caused by two of the five animals being induced and the other three not. This heterogeneity is because these female littermates of Tfm/Y animals are of two genotypes: Tfm / + and + / + . The Tfm / + mice have two populations of cells in their kidneys, one ex- presses only the Tfm gene and the other the + gene due to random X-chromosome inactivation (Lyon, 1972). It is the Tfm expressing cells in Tfm/+ mice that probably produce the part-induction phenotype in some + / ? littermates of Tfm/Y animals (Table 2).

The heterogeneity due to random X-inactivation in Tfm/+ mice is further illustrated by the marked asymmetry of induction of H D C by testosterone between left and right kidney in some of the + / ? female littermates of Tfm/Y animals compared with wild-type, + / + animals (Table 3; animals 6, 7, 8 and 10 are probably Tfm/+ and animals 9, 11 and 12, + / + ) .

Table 2. The effect of testosterone on female littermates of Tfm/Y males in kidney HDC levels

Testosterone Phenotype Control (N) implanted (N)

C57BL/10 +/+ 0.30 + 0.08 (3) 0.047 _+ 0.009 (3) (normal females) C57BL/10 +/? 0.32 + 0.05 (5) 0.130 + 0.033 (5) (female littermates of Tfm/Y animals)

Steroid regulation of enzymes in Tfm/Y mice

Table 3. HDC activity in single kidneys of individual testosterone implanted littermates Tfm/Y and control females

of

Animal Left Right Strain no. Implant kidney kidney

C57BL/10 +/+

C57BL/10 +/+

C57BL/10 +/? (female littermates of Tfm/Y animals)

1 None 0.421 0.413 2 None 0.382 0.361 3 None 0.439 0.425 4 Testosterone 0.003 0.004 5 Testosterone 0.001 0.002 6 Testosterone 0.131 0.153 7 Testosterone 0.220 0.049 8 Testosterone 0.201 0.042 9 Testosterone 0.003 0.019

10 Testosterone 0.602 0.439 11 Testosterone 0.002 0.014 12 Testosterone 0.001 0.005

223

The effect of differing levels of ~-oestradiol on HDC induction in C57BL/lO female mice

If the induction of HDC by testosterone in Tfm/Y animals is due to aromatisation of testos- terone to produce fl-oestradiol, it should be possible to mimic this effect in normal C57BL/10 females. It is known, however, that implants of fl-oestradiol do not produce such a large induction as that ob- served in Tfm/Y animals implanted with testosterone (Table 1). It is possible that either another oestrogen is responsible for the Tfm/Y response, or that the dose of fl-oestradiol from the implants is either too large or too small to produce the response in normal females. To test the first possibility, oestrone and oestriol were pelleted and implanted into normal C57BL/10 females but neither produced such high HDC levels as in testosterone implanted Tfm/Y animals (1.59 and 0.31 units, Table 4 cf. 5.97 units Table 1). Oestriol had no effect on HDC levels, although oestrone proved a potent inducer, being more effective than fl-oestradiol itself (Table 4).

Thirty-milligram pellets of fl-oestradiol produce circulating levels of over 100 pg/ml compared with physiological levels of approximately 2pg/ml (R. Webb, personal communication) therefore two methods of supplying smaller doses of fl-oestradiol than the cellulose implants were tried. The first was to inject a single dose of fl-oestradiol daily, either 10pg (i.e. roughly the physiological level of oestra- diol in the mouse) or 100 pg. The second method was to implant two different lengths of silastic tubing filled with fl-oestradiol; Butterstein et al. (1980) have shown that silastic tubing implants produce a slow release of fl-oestradiol in the mouse.

These alternative methods of fl-oestradiol adminis-

tration produced a greater induction of HDC than the pellets (Table 4), the most successful treatment was the silastic implants which induced H D C to levels approaching those found in testosterone im- planted Tfm/Y animals (4.06 and 3.40 cf. 5.97 units, Tables 4 and 1).

DISCUSSION

In normal mice, testosterone represses kidney histi- dine decarboxylase levels and fl-oestradiol induces them (Kahlson and Rosengren, 1968; Mart in et al., 1984). Tfm/Y mutant mice respond abnormally to testosterone; HDC is induced over 1000-fold to levels surpassed only by thyroxine induction (Table 1). These results confirm the previous report of testos- terone induction of HDC in Tfm/Y mice (Bulfield and Nahum, 1978) where it was suggested that the phenomenon might be caused by aromatisation of testosterone to oestrogens, due to the absence of the kidney cytosol androgen receptor (Gehring and Tomkins, 1974).

This hypothesis has been tested in three ways: (a) the effects of both aromatisable and non- aromatisable androgens on kidney HDC levels in TfmfY mice; (b) the induction of HDC in normal female mice by different oestrogens and varying doses of fl-oestradiol, and (c) the effect of testosterone on kidney HDC levels in Tfm/+ female mice which have a mixed populat ion of Tfm and + type cells in their tissues.

Kidney HDC levels in Tfm/Y mice were not in- duced by non-aromatisable androgens (5~-dihydro- testosterone and 5ct-androstane-3,17-dione) but they are also not induced by androstene-4-ene-3,17-dione

Table 4. The effect of various oestrogen treatments on HDC levels in normal C57BL/10 females Significance

(P) of difference Method of HDC from "control"

H o r m o n e administration activity (N) (t -test) Control - - 0.30 _+ 0.08 (6) - - fl-Oestradiol pellet implant 0.60 + 0.20 (4) <0.01 Oestrone pellet implant 1.59 + 0.40 (3) <0.001 Oestriol pellet implant 0.31 _+ 0.11 (3) NS fl-Oestradiol 10 pg injected daily 1.72 _+ 0.26 (5) < 0.01 fl-Oestradiol 100pg injected daily 1.38_+ 0.14 (5) <0.01 fl-Oestradiol silastic implant (0.5 cm) 4.06 _+ 0.53 (6) < 0.01 fl-Oestradiol silastic implant (1.0 cm) 3.40 _ 0.54 (5) <0.001

224 RICHARD J. MIDDLETON and GRAHAME BULFIELD

(Table 1) which has been shown to have identical aromatisable properties to testosterone in rat brain (Parrott, 1975; Selmanoff et al., 1977). It is possible that either, androstene-4-ene 3,17 dione is not aro- matisable in the mouse kidney or, that some other mechanism is responsible for the testosterone in- duction of HDC in Tfm/Y mice. Kidney ornithine decarboxylase levels in Tfm/Y mice behave in the expected manner for an androgen-inducible enzyme: they are not induced by any of the four androgens tested (Table 1). The only unusual effect is that ODC in mouse kidney is not inducible by fl-oestradiol in contrast to its induction in rat kidney (Table 1 and Nawata et al., 1980).

If aromatisation of testosterone to oestrogens is the cause of kidney HDC induction in Tfm/Y mice, then it should be possible to produce quantitatively the same level of HDC induction by oestrogen adminis- tration. Thirty-milligram pellets of fl-oestradiol do not produce such large levels of HDC induction (0.35 units cf. 5.97 units; Table 1); neither do 30 mg pellets of two other oestrogens, oestrone and oestriol (1.59 and 0.31 units respectively, Table 4). As such high levels of oestrogens could cause desensitisation or down regulation of receptors, lower doses of fl-oestradiol were administered, which produced higher levels of HDC induction. The greatest in- duction was achieved with 0.5 cm silastic implants (producing small regular increases in circulating hor- mone levels; Butterstein et al., 1980) which induce kidney HDC levels to 4.06 units compared with 5.97 units in testosterone implanted Tfm/Y animals (Tables 4 and 1). Oestrogens are therefore capable of producing the large kidney HDC levels seen in Tfm fY mice.

The interaction of testosterone with the Tfm gene product and the Hdc gene was examined in a different way. Female littermates of Tfm/Y males are of two genotypes, + / + and Tfrn/+; the Tfm/+ animals are chimaeric and have two populations of cells, Tfm and -I-, caused by random X-chromosome inactivation (Lyon, 1972). If aromatisation of testosterone to oestrogens occurs within the kidney cells of Tfm/Y mice, then in Tfm/+ animals, the Tfm ceils should be producing oestrogens and hence produce induction of HDC and the + cells should bind testosterone normally to its receptor and hence produce repression of HDC. Therefore Tfm/+ animals should display widely varying phenotypes after testosterone im- plantation depending on the proportion of Tfm:+ genes inactivated in their kidney cells. Individual animals should also vary in phenotype between left and right kidneys. Both these phenomena are ob- served in +/? female littermates of Tfm/Y mice (Tables 2 and 3); after testosterone implantation putative Tfm/+ animals have relatively high HDC levels in at least one kidney (Table 3; animals 6, 7, 8 and 10; average 0.292 units) and putative + / + animals have low HDC levels (Table 3; animals 9, 11 and 12; average 0.019 units). Three of the four putative Tfm/+ mice (7, 8 and 10) also show asym- metry in HDC levels between left and right kidneys. These results suggest that aromatisation of testos- terone to oestrogens in Tfm/Y mice takes place in individual kidney cells where the oestrogens induce the Hdc gene.

We conclude that, androgen-induction of kidney HDC in Tfm/Y animals is a characteristic of testos- terone and not of other aromatisable and non- aromatisable androgens, can be mimiced in normal females by administration of low doses of fl-oestra- diol and, is a function of individual kidney cells. While all this evidence points to the direct aro- matisation of testosterone to oestrogens in the kidney cells of Tfm/Y mice the final proof will have to await the direct analysis of this transformation in cells from Tfm/Y and + / + animals.

Acknowledgements--We would like to thank Kathy Will- iamson for technical assistance and Drs David Armstrong, Bob Webb and Sam Martin for helpful discussions.

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