Cytosol and nuclear levels of thymic progesterone receptors in pregnant, pseudopregnant and steroid-treated rats

J. sreroid Biochem. Vol. 25, No. I, pp. 65-69, Printed in Great Britain. All rights reserved

1986

0022-4731186 $3.00 + 0.00 Copyright C 1986 Pergamon Journals Ltd

CYTOSOL AND NUCLEAR LEVELS OF THYMIC PROGESTERONE RECEPTORS IN PREGNANT, PSEUDOPREGNANT AND STEROID-TREATED RATS PAUL

PEARCE

and

JOHN W.

FUNDER*

Medical Research Centre, Prince Henry’s Hospital, Melbourne 3004, Australia (Received 25 February 1985)

Summary-Cytosol and nuclear levels of progesterone receptors in rat thymus and uterus were compared in mature Sprague-Dawley rats under a variety of physiological (non-oestrus, oestrus, pregnancy) and experimental circumstances (pseudopregnancy, oestrogen administration, oestrogen plus progesterone administration). Cytosols and nuclear extracts were charcoal treated to remove endogenous steroids, and incubated overnight at 4°C with the synthetic progestin [3H]R5020 _+ > IOO-fold R5020, in the presence of > 200-fold RU26988 (a highly specific synthetic glucocorticoid) to exclude tracer from glucocorticoid receptors. In the various states examined, fevels of progesterone receptors in the uterus were an order of magnitude higher (35&1300 fmol/mg protein) than in the thymus (40- 140 fmol/mg protein). fn contrast, considerable parallelism between uterus and thymus was seen in terms of the effects of the various manipulations upon tissue levels of progesterone receptors, and of their distribution between cytosol and nuclear compartments.

that of pregnant animals, and though the conceptus is absent the corpus luteum persists. Pseudopregnancy lasts for 12-13 days[ll, 121, over which period low oestradiol levels are found accompanied by elevated progesterone, though the latter falls n,pidly at the end of pseudopregnancy [13-151.

INTRODUCTION

In previous studies we have demonstrated the presence of putative progesterone receptors (PR) in male and female rat thymus [l]. Though the function of these receptors remains obscure in the male, a possible role in the female may relate to thymus-related immunosuppression during pregnancy [Z-6]. Pregnancy is characterized by prolonged elevation of plasma progesterone levels; both progesterone and oestradiol have been reported as determinants of the immunosuppression of pregnancy [7]. By contrast, the elevated glucocorticoid levels of pregnancy do not appear to be directly involved, on the basis of studies demonstating differences between the effects on the thymus of pregnancy and glucocorticoid treatment [6]. Similarly, studies on the effect of pregnancy plasma on mixed leucocyte cultures showed no correlation between level of plasma glucocorticoid and inhibition of thymidine uptake [8]. The presence of PR in the thymus suggests that direct effects of progesterone on the thymus may occur during pregnancy. The present study thus was undertaken to further characterize the regulation of thymic PR during pregnancy. Levels of thymic cytosol and nuclear PR were examined at a fixed time point of pregnancy (day 12), and thymic levels compared with those in the uterus. As controls we have used normal cycling rats, and pseudopregnant rats previously shown to be appropriate additional controls in pregnancy-related studies [9, lo]. Pseudopregnant animals have a hormonal profile similar to *To whom correspondence

EXPERIMENTAL

Virgin female Sprague-Dawley rats were obtained at 45-50 days of age from the Central Animal House, Monash University. The animals were kept on a 12 h light-12 h dark cycle (lights on 0600) and were fed ad libitum on rat chow (GR2 pellets, Clark King, Melbourne, Australia). Vagina1 smears were examined by microscopy to determine the stage of the oestrous cycle; late pro-oestrous to early oestrous rats were chosen for mating studies. Animals were mated with adult males (1 male per 2 females), with day 1 of pregnancy taken as the day the plug was found. For induction of pseudopregnancy animals of a similar oestrous stage were stimulated vaginally for a few seconds using a smooth wooden swab. This manoeuvre was deemed day 1 of pseudopregnancy. Animals were killed by decapitation, and blood collected into tubes containing heparin, centrifuged, and the plasma frozen for steroid analysis. The thymus and uterus of each animal was placed in iced normal saline, blotted, weighed and transferred to tubes containing buffer [ 10 mM Tris, 1.5 mM EDTA, 2mM dithiothreitol (DTT), 10mM sodium molybdate, and 10% glycerol (TEDMG); glycerol and DTT were added fresh daily]. Tissues were homogenized by short bursts of a Polytron PlO (Kinematica GMBH,

should be addressed. 65

66

PAUL PEARCEand

Luzern, Switzerland) at speed 3. The temperature of homogenization was maintained low by means of an ice bath, and where more than one 5 s burst was needed, a 10 s interval was allowed for cooling. Homogenates were centrifuged at 8OOg for 10 min (Sorval RC5, DuPont-Biomedical Division, Newtown, Conn.), and the supernatant then centrifuged at 100,OOOg for 1 h in a Kontron TGA65 ultracentrifuge (Kontron International, Zurich, Switzerland), for cytosol. The 8OOg pellet was washed twice with TEMDG and then extracted with 0.6 M KC1 in TEDMG for i h at 4°C with intermittent vortexing. Extracted samples were centrifuged at 100,OOOg for 30 min in the TGA 65, with the supematant taken as a crude nuclear extract. The cytosol and nuclear extract were treated with charcoal, to remove free steroid and other substances shown to affect tracer binding [ 161. An equivalent volume of dextran-charcoal(O.O5% : 1% in TEDMG) was pelleted, the buffer decanted and the cytosol or nuclear extract added. The charcoal was resuspended by vortexing, and the mixture allowed to stand for 10 min. The tubes were then centrifuged at 4000 g in a HS4 Sorvall rotor for 10 min, and the supernatant fractions used for receptor analysis. Receptor analysis was carried out by incubating 0.2ml of supernatant with 0.2ml of [3H]R5020 (Promegestone; DuPont-NEN, Boston, Mass.) at a single saturing dose (10 nM). Non-specific binding was determined by incubating duplicate tubes in the presence of 1 pm radioinert R5020. All tubes contained 1% ethanol, and 200-fold excess RU26988 (1 l&l 7/$dihydroxy- 17-( l-propynyl~-androsta1,4,6trien-3-one; gift of Roussel-Uclaf, Paris, France) to block [‘H]R5020 binding to glucocorticoid receptors. Incubation was termined after 20 h at 4°C by the addition of an equal volume of 0.05%:0.5% dextran charcoal in TEDMG buffer. Tubes were twice shaken over a 10min period, centrifuged at 4000g for IOmin, and the supernatant counted in lOm1 of aqueous scintillant (5.5 g PPO, 0.1 g POPOP, 333 ml Teric X10,667 ml toluene) in a Packard Tricarb 46OC

JQHN W. FIJNDER

Table

1. Thymus

weight, body weight and physiological

state

Physiological

state (a) Pregnancy (17) Pseudopregnancy (I 5) Non-oestrus (26)

Oestrus(8)

Thymus

Body

(mg)

(g)

614+25 619 + 36 638 f 29 643+52

202 i 6 17358 153+5 181 + I4

Thymus/Body

(mf#OOs) 304 359 417 355

i: 22 i_ 38 j: 34 i: 61

scintillation counter with dpm program (Packard Inst~ments Co., Downers Grove, Ill.). Protein concentration was estimated in each sample using the Bradford Coomassie Blue method 1171. In those experiments involving the administration of steroid, 0.2 ml of oestradiol benzoate (E,B; 4 p g) in maize oil, or 0.2 ml of oil, was administered i.m. daily for 2 days. One hour before sacrifice, either vehicle, or 1 mg of progesterone (P) as a suspension in 0.5 ml normal saline, was administered IP. The animals were killed and bled as described above. Levels of oestradiol and progesterone were determined in plasma using WHO reagents by a previously described method [IS]. RESULTS

Changes in thymic weight us physiological state

Table 1 shows the mean body weight and thymus weights (*SE) of animals used in this study. There is no significant difference (Student’s r-test) between any group in terms of thymus weight. The pregnancies were syngeneic, and our previous studies have yielded similar findings 111. Translocation of cytosol receptors To validate the measurement of cytoplasmic and nuclear PR by exchange assay, rats were injected with oil, oestradiol benzoate (E,B) or E,B followed by progesterone (P), and fractions prepared as detailed in Experimental. Results of these preliminary studies are shown as Fig. 1. In oil-injected rats with intact ovaries, the preponderance of PR in both thymus (left

Thymus

Fig. 1. ComDartmentalization of Drosesterone receptors in cytosol (open bars) and nuclear extracts (hatched) of ihymus (left panel) and uterus (right panel). Rats used we;e intact,’ randomly cycling and injected i.m. with 0.2 ml maize oil (Control), 4 pg oestradiol benzoate daily for 2 days (E,B), or 4 @g oestradiol benzoate daily for 2 days followed by 1 mg progesterone 1 h before sacrifice. Values shown represent the mean + SE, n = 4.

Regulation of thymic progesterone receptors Table 2. Effect of charcoal treatment on specific binding of [‘H]R5020 in cytosol and nuclear fractions from non-oestrous rat tissues Cytosol Organ (n) uterus (5) Thymus (5)

Nucleus -c

+c

+c

-c

370 + 80 225 + 30 120 k 20 100 + 10 27 + 9 14+ 1 12*2 18k4

Values given are fmol/mg, mean k SEM; +C charcoal pretreated, -C no pretreatment.

panel) and uterus (right panel) is in the cytoplasmic compartment. In rats injected with E,B, the total level of PR increases, but the cytoplasmic predominance persists, in both tissues. In rats injected with E,B plus P, total PR levels are equivalent to those in rats injected with E,B alone; there is, however, a marked intracellular redistribution, with the predominance of PR now in the nucleus. Charcoal treatment of cytosol and nuclear extracts

The data in Fig. 1 were obtained from cytosols and nuclear extracts treated with dextran-coated charcoal before receptor assay, to ensure that high levels of steroid present in some of the samples did not interfere with the receptor assay. Koseki et a1.[16] have demonstrated the advantages of such a step for analysis of PR, to remove not only endogenous steroid but also other inhibitory factors. Preliminary studies (Table 2) comparing non-treated and charcoal-pretreated preparations showed very little difference in level of nuclear PR, but consistently higher levels of cytoplasmic PR in treated samples, regardless of the measured level of plasma progesterone in the animals. Nuclear and cytosol PR in thymus and uterus

Between thymus and uterus, there is an overall pattern of similarity in the intracellular distribution of PR between cytosol and nucleus, in any of the reproductive situations examined (Fig. 2). In terms of PR, the main difference between the thymus and uterus is the much higher uterine PR levels. Since, however, levels of PR are much higher in the thymic

r 1 Thymus

67

epithelioid cells than in T-cells [l], this marked apparent difference in levels (in terms of total tissue protein) may be much less in terms of sites per target cell. In both tissues, however, there are marked differences between reproductive states in terms of the ratio of nuclear to cytosol PR concentration; in pregnant animals, for example, this ratio approaches unity. This pattern appears common to animals with high levels of progesterone (as detailed below), being even more marked in animals treated with E,B + P, though such animals have higher total levels of PR, presumably due to the prior administration of E,B. The absolute concentration of nuclear receptors in pregnant animals is the same as in oestrus and pseudopregnancy, but higher than in non-oestrus. This is true for both uterus and thymus; thus, while the nuclear to cytoplasmic ratio varies between oestrus, pregnancy and pseudopregnancy, the concentration of nuclear receptor remains fairly constant. Pseudopregnancy appears to be equivalent to oestrus both in terms of PR concentrations and the distribution between cytosol and nucleus. It thus appears to be quite distinct from the profile seen in pregnancy. In pseudopregnancy, as opposed to oestrus, the animal has been subjected to high P levels for _ 11 days, and a short period of declining levels of P, in the absence of high levels of oestrogen. Steroid levels in plasma

Since oestrogen and progesterone have both been shown to affect the level of progesterone receptors [19-231, plasma levels of each steroid were determined and are shown in Fig. 3. Except when animals were given E,B, the levels of E, were at or below levels of detection (upper panel). A few of the pseudopregnant animals showed values above the detection level (0.08 nM); these may represent animals starting to go into oestrus, although examination of the ovaries indicated the rats to be still pseudopregnant. Progesterone levels are shown in the lower panel. Levels are not significantly different between groups with two exceptions, the pregnant and progesterone-

I 1.2

I

T

Non._ oestrus Pregnant P‘seudo ___._

Fig. 2. Compartmentalization

I

1 fn ml

Pseudo

pregnant

of progesterone receptors in cytosol (open bars) and nuclear extracts (hatched) of thymus (left panel) and uterus (right panel). Rats used were not in oestrus, as defined by vaginal smear (Non-oestrus), in the oestrous state (Oestrus), 12 days pregnant, or at day 11-12 of pseudopregnancy (PsP). Values shown are mean k SE, n inset.

PAUL PEARCE and

68

NE

E

P

PSP

c

NE

E

P

PSP

c

E,B

E,E+P

r

0

JOHN W. FUNDER approximately one. This indicates that during pregnancy, in contrast with the other states examined, a higher percentage of receptors are activated and nuclear bound. The low cytosol levels of PR may reflect the low oestradiol levels of pregnancy; alternatively they may mirror a relatively high synthesis and turnover of active receptors, but yielding a lower steady state. The possible role of the PR and progesterone in thymic involution and weight loss observed in some studies on pregnancy highlights a species difference. Numerous studies with pregnant mice have demonstrated thymic weight loss and involution during pregnancy [2,3,28]. Chambers demonstrated this effect in both allogeneic and syngeneic matings [IO]. In rats, however, Forster showed that pregnancy following syngeneic matings produced no changes in thymic weight, but those following allogeneic matings led to increased thymic weight; pseudopregnant rats showed decreased thymic weight [9]. Our own study has confirmed the lack of change in thymic weight in syngeneic pregnant rats, but not the previous findings in pseudopregnant rats. In previous studies mice given 5 mg of progesterone for 7 days showed no significant change in thymic weight or thymic cell number [29]. The thymic involution and weight loss observed in some

!a 16)

16)

:,!3

E@+P

Fig. 3. Plasma levels of oestradiol (top panel) and progesterone (bottom panel) in rats under various conditions. NE = non-oestrus, E = oestrus, P = pregnant, PsP = pseudopregnant, C = random cycling controls, E,B = oestrogen injected, E,B + P = oestrogen and progesterone injected. Values shown are mean + SE, n inset.

treated animals. These two groups have similarly high levels, 2-3 times those of other groups. The levels seen in pseudopregnancy are to be expected at this stage, representing a fall from the higher levels usually seen on day 11. DISCUSSION

The regulation of uterine progesterone receptors by oestrogen has been well documented. Low cytosol levels are found in the absence of oestrogen, or after the administration of progesterone; in the latter instance, there is a concomitant rise in nuclear progesterone receptor levels [19-241. This regulation by oestrogen occurs in the thymus as in the uterus. The measurement of increased receptor levels in the nuclear compartment is consistent with current models of steroid-receptor action, whether or not unoccupied progesterone receptors are initially nuclear, as has been shown for oestrogen receptors [25,26] and reported for progesterone receptors [27]. The existence of regulated progesterone receptors in the thymus is consistent with the possibility of direct effects of progesterone on the thymus during pregnancy. The ability of the thymus to maintain fairly constant levels of nuclear progesterone receptors in pregnancy is a feature in common with the uterus. Similarly, in pregnancy, both tissues show a ratio of nuclear to cytosol progesterone receptors of

studies [2,3,27] therefore appears to be more complex than a simple direct effect of progesterone on thymocytes. Progesterone is a predominant or complete glucocorticoid antagonist [30, 311, and also capable of displacing glucocorticoids from transcortin, the plasma binding globulin; a balance between these activities may therefore be involved in the effects of progesterone upon thymic weight and involution. Androgens and oestrogens have an effect similar to glucocorticoids on thymic lymphocytes-albeit an indirect one, since receptors for these steroids are in epithelioid cells and not thymocytes [29]. Grossman et al. have reported putative oestrogenand androgen-dependent blood borne proteins of thymic origin, which he has suggested may be responsible for the immune effects seen after treatment with oestrogens or androgens [32, 331. It is possible that a similar factor may be produced by the thymus in response to progesterone, and may be responsible in part for the immunosuppressed state of pregnancy.

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