Enzyme activities in the androgenized rat uterus refractory to oestrogenic stimulation

J. steroid Biochem. Vol. 25, No. 4, pp. 491496,

1986

0022-4731/86

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ENZYME ACTIVITIES IN THE ANDROGENIZED RAT UTERUS REFRACTORY TO OESTROGENIC STIMULATION Z. SCHWARTZ*, J. F. GUEST, M. G. ELDER and J. 0. WHITE? Institute of Obstetrics and Gynaecology, Hammersmith Hospital, Ducane Road, London WI2 OHS, England (Received 23 April 1986)

Summary-Uterine enzymes involved in the intermediary metabolism of glucose have been measured in the androgenized rat in which there is evidence of diminution of the oestrogenic responses despite raised glycogen and glucose typical of maximal oestrogenic stimulation. Phosphofructokinase and isocitrate dehydrogenase (NADP, cytosolic) activities were significantly decreased in the androgenized rat and were elevated following treatment with natural progesterone and synthetic progestins which partially reverse the uterine abnormalities of the androgenized rat. Mitochondrial protein was decreased in the uterus of the androgenized rat but there was an apparent sparing effect on isocitrate (NAD) and malate (NAD) dehydrogenase. The data suggest that selective effects on specific enzymes involved in intermediary

metabolism are a feature of the refractory state associated with constant oestrogenic stimulation. The possible cellular mechanisms underlying these effects are discussed.

The activities of cytosolic isocitrate dehydrogenase (NADP) and malic enzyme (NADP) which are important sources of reducing equivalents for biosynthetic processes were also measured. Enzymes that were found to be changed in the androgenized rats were further studied following treatment with natural and synthetic progestins, which previously have been shown to partially restore the capability of the uterus to respond to oestradiol[9, lo].

INTRODUCTION

neonatal administration of testosterone propionate to female rats induces a syndrome of adult sterility (androgenization) characterised by failure of ovulation and corpus luteum formation and a constantly cornified vaginal epithelium [ 1,2]. Such adult anovulatory rats have plasma oestradiol concentrations in the range found during the morning of proestrus [3] whereas progesterone is low or undetectable [4]. Continuous oestrogenic stimulation results in the uterus becoming refractory as evidenced by decreased synthesis of the oestrogen-induced protein (IP) [S], and progesterone receptor [6] and an

The

EXPERIMENTAL Chemicals

All chemicals for enzyme assays were purchased from the Sigma Chemical Co., Poole, Dorset, England. All other chemicals were obtained from BDH Chemicals Ltd, London, England and were of Analar grade.

attenuation of the growth response [7, 81. Despite this evidence of a diminished response to oestrogen, glycogen content is raised and is typical of the uterus responding maximally to oestradiol[7,8]. Uterine

glucose concentration is also elevated in androgenized compared with normal rats [7]. It was of interest therefore to determine if the enzyme activities involved in the intermediary metabolism of glucose to provide precursors and energy for macromolecular biosynthesis were decreased in the uterus of the androgenized rat. Uterine enzyme activities were compared in control and androgenized uteri and included: glucose-6-phosphate dehydrogenase (G-6PD) the first enzyme of the pentose phosphate pathway, pyruvate kinase and phosphofructokinase rate limiting enzymes of glycolysis, isocitrate and malate dehydrogenases (NAD+, mitochondria) the former being a rate limiting enzyme of the citric acid cycle.

Production of adult anovulatory rats

Female wistar rats were used throughout. Five-day old rats were injected with testosterone propionate (1.2 mg/animal) as previously described [6] and when adult (older than 120 days) were used for measurement of uterine enzyme activities. Control animals of similar age were selected at random from a colony of normal females derived from the same breeding stock. Previous reports have indicated that elevated glycogen in the uteri of androgenized rats is observed in comparison both with random cycling rats [7] and those selected at dioestrus and oestrus [8]. Progestin and tamoxifen treatment of anovulatory rats

*On Study Leave from Kaplan tTo

whom

correspondence

Hospital, Rehovot, should be addressed,

Animals received either (a) Progesterone (pregn-4ene-3,20-dione; Sigma Chemical Co.) (b) norgestrel

Israel.

491

492

Z. SCHWARTZ et al.

[(+)-13~-ethyl-17-hydroxy-18,19-dinor-17cc-pregn4-en-20-yn-3-one; Schering AG, Berlin/Bergkamen, Germany] (c) norethisterone acetate (17a-ethynyl17/I-hydroxy-3-oxo-oestr-4-ene 17/I-acetate; Sigma Chemical Co. Ltd) (d) medroxyprogesterone acetate (17 acetoxy-6-methyl-4-pregnene-3,20-dione; Upjohn Ltd, Crawley, W. Sussex) each for 6 days at a dose of 1 mg/day. Tamoxifen (ICI 46474) was administered on 4 consecutive days at a dose of 500 pg/day. All steroids were administered subcutaneously in propylene glycol (0.2 ml) and animals were killed 2426 h after the last injection of progestin or tamoxifen. Subcellular fractionation

of uteri

Uterine tissue from three animals was pooled to form one group and all subsequent operations were performed at 4°C. The uterine horns were minced and homogenised as previously described [6] in 5 vol of 0.32 M sucrose; 20 mM phosphate, pH 7.4; 2 mM EDTA. The homogenate was centrifuged at 1OOOg for 10min to remove the nuclear pellet and the supernatant recentrifuged. The resulting supernatant was centrifuged at 14,000 g for 30 min to yield a crude mitochondrial pellet. The post-mitochondrial supernatant was centrifuged at 100,OOOg for 1 h to produce a soluble cytosolic fraction. The crude mitochondrial pellet was purified by centrifugation through a 1.2 M sucrose/phosphate buffered cushion at 60,000 g for 2 h and the resulting pellet was washed by resuspension in 0.32 M sucrose/phosphate buffer followed by centrifugation.

5 nM MgCl,; 2mM phosphoenol-pyruvate;

ADP; 0.3 mM NADH; 2 IU of LDH.

1 nM

Phosphofructokinase (PFK) PFK activity in cytosol was measured in 50 mM Tris-HCl, pH 7.5 containing 1 mM ATP; 2 mM D-fructose-6-phosphate; 1 mM phosphoenolpyruvate; 0.25 mM NADH; 50 PM di-adenosine pentaphosphate; 2mM AMP; 1 IU of pyruvate kinase; 2 IU of lactate dehydrogenase. Isocitrate dehydrogenase (ICDHINADP) ICDH (NADP) activity in cytosol was measured in 40 mM Tris-HCl, pH 7.2 containing 0.72 mM 0.7 mM ADP; 0.2 mM NADP; 64 mM Mn Cl,; DL-iSOCitriC acid. Isocitrate dehydrogenase (ICDHINAD) Mitochondrial ICDH(NAD) activity was measured in 35 mM Tri-HCl, pH 7.2 containing 0.72 mM MnCl,; 0.7 mM ADP; 0.7 mM NAD; 0.1% Triton X- 100; 64 mM DL-isocitric acid. Malate dehydrogenase (MDHINAD) MDH(NAD) Mitochondrial activity was measured in 1OOmM phosphate buffer, pH 7.4 containing 0.25 mM NAD; 2mM L-malic acid; 0.5% Triton x-100. DNA and protein estimations These were determined by the procedures Burton[ 1 l] and Lowry et a/.[ 121 respectively.

of

Enzyme assays

Statistical analysis

Enzyme activity was assayed spectrophotometrically (Gilford 250 spectrophotometer) at 340 nm which measured the change in absorbance due to a change in either NADH or NADPH. All enzyme assays were performed at 30°C in a final volume of 1 ml.

Tests of significance between enzyme activities was performed using two tailed Student’s t-test. All values are mean f SD, where n = the number of separate determinations.

Lactate dehydrogenase (LDH) LDH activity in cytosol was measured in 100 mM phosphate buffer, pH 7.4 containing 0.25 mM NADH; 2 mM pyruvic acid. Glucose 6-phosphate dehydrogenase (G 6-PD) G-6PD activity 160mM Tris-HCl, o-glucose-6-phosphate; MgClz.

in

cytosol was measured in pH 7.5 containing 1 mM 0.4 mM NADP; 8mM

RESULTS

There was no significant difference in the protein concentration of the cytosol fractions from uteri of control, androgenized and progesterone treated androgenized rats. The protein concentration of the mitochondrial fraction from androgenized uteri was however significantly lower than control and was significantly increased following progesterone treatment (Table 1). There was no significant difference in the protein: DNA ratio in each of the treatment groups.

Malic enzyme [MDH (NADP)]

Cytosolic enzyme activities

MDH (NADP) activity in cytosol was measured in 100 mM phosphate buffer pH 7.4 containing 2 mM r.-malic acid; 0.25 mM NADP.

The specific activities of phosphofructokinase and glucose-6-phosphate dehydrogenase were each significantly decreased in androgenized compared with control rats (Table 2) as were NADP-isocitrate dehydrogenase and malic enzyme. There was no significant difference in the specific activities of pyruvate kinase and lactate dehydrogenase between

Pyruvate kinase (PK) PK activity 50 mM Tris-HCl,

in cytosol was pH 7.5 containing

measured in 50 mM KCl;

Uterine

Table

1. Cytosol

enzyme

activities

and mitochondrial

493

protein

content

Control

Androgenized

Progesterone

Cytosol wet weight mg/mg DNA

* 3.94 14.71 * 2.42

+ I .46 15.76 + 3.22

35.75 f 2.65 17.23 f 2.71

Mitochondria mg/g wet weight mg/mg DNA

I .Ol + 0.22 0.38 + 0.11

0.29 * 0.141 0.12 * 0.07’

0.44+0.10f 0.21 + 0.057

Protein concentration of cytosol and mitochondrial fractions prepared from uteri of control, androgenized and progesterone treated androgenized rats as described in Experimental. Values are mean f SD (n = 8, control; n = 9, Androgenized, Progesterone). Statistical analysis was by Student’s r-test (two-tailed). Significantly different from control: ‘P < 0.005. Significantly different from androgenized: tP < 0.01; JP < 0.02.

control and androgenised rats. Expression of each enzyme activity relative to DNA revealed the same pattern of change (data not shown).

drogenase (NAD) was also increased in androgenized compared with control rats, however, in contrast to ICDH (NAD) the activity of MDH (NAD) relative to DNA was decreased.

Mitochondrial enzyme activities

Effect of progesterone on cytosolic enzymes

There was minimal contamination of mitochondrial preparations by cytosolic components as assessed by the undetectable activity of lactate dehydrogenase. The specific activity of isocitrate dehydrogenase (NAD) was significantly increased in the uteri of androgenized rats although there was no significant difference from control when expressed relative to DNA. (Table 3). This therefore would suggest that ICDH activity is being maintained against a loss of other mitochondrial protein. There were no obvious ultrastructural changes in the mitochondria prepared from uteri of androgenized rats (Ryder, Guest and White-unpublished observations). The specific activity of uterine malate dehy-

The specific activities of PFK and ICDH (NADP) were increased following treatment of androgenized rats with progesterone, norgestrel, norethisterone and medroxyprogesterone acetate (Fig. 1). Tamoxifen did not significantly affect PFK, ICDH (NADP), G-6PD or malic enzyme. The specific activity of G-6PD was increased following progesterone while that of malic enzyme was increased only in response to norethisterone. Effect of enzymes

treatment

on

The specific activity of ICDH

Table 2. Cytosol

PFK (nmol NADH used) PK (nmol of NADH used) G-6-PD (nmol of NADPH produced) LDH (nmol of NADH used) ICDH (nmol of NADPH produced) MDH (nmol of NADPH produced)

progestin

(NAD) was de-

enzyme activity

Control

Androgenized

0.95 f 0.38 (15) 0.19~0.04(15) 33.38 k 5.62 (10) 0.26 f 0.09 (12) 24.71 *4.54(15) 3.62 f 1.07 (15)

0.49iO.ll (15) 0.17+0.06(15) 26.58 f 6.33 (10) 0.28 f0.17(13) 15.07k6.30(13) 2.64 * 0.88 (15)

Significance

P P P P P P


Enzyme activities measured in the cytosol fractions prepared from the uteri of control an androgenized rats. Assays were performed as described in Experimental. The results are mean ?r SD of the number of determinations indicated in parentheses and are expressed per minute per mg protein. The significance of the difference in each enzyme between control and androgenized groups is shown and was derived from unpaired Student’s f-test (two-tailed). Table 3. Mitochondrial

enzvme activities

Control

Androgenized

Progesterone

86.54 + 16.97* I .07 * 0.43 (9)

42.42 f 9.435 0.88 k 0.26 (9)

ICDH (Activity/min/mg (Activity/min/mg ” MDH (Activity/min/mg (Activity/min/mg n

protein) DNA)

25.06 i 8.90 1.36 + 0.61 (8)

protein) DNA)

54.99 t 9.24 4.67 + I .94 (5)

114.12 i 33.30t 2.17 f 0.58t (6)

mitochondrial

80.69 + 17.76 2.67 k 0.88 (6)

Enzyme activities measured in the uterine mitochondrial fractions of control, androgenized and progesterone-treated androgenized rats. The activities (nmoles NADH produced) are the mean f SD of the number of determinations indicated in parentheses. Statistical analysis was by unpaired Student’s r-test (two-tailed). Significantly different from control: 'P < 0.001; tP < 0.005; $P c 0.025. Significantly different from androgenized; $P c 0.001. Tables I, 2 and 3: Values are mean f SD.

494

Z. SCHWARTZ et al.

50

ICDH (NADP)

G-

6PD

(Cl

40 Ial

Lbfm

30 f 6

20

ii m E

10

’

0

E 1

6

;

5

PFK

MDHtNADPI

1

0 C

A

P

T

Ng

Nf

Mp

c

A

P

T

Ng

Nr

Mp

Fig. I. Enzyme activities following hormone administration to androgenised rats. The enzyme activities of phosphofructokinase (PFK), malic enzyme (MDH, NADP), isocitrate dehydrogenase (ICDH, NADP) and glucose-6-phosphate dehydrogenase (G6PD) were measured in the uteri of androgenized rats (A) and following progesterone (p), tamoxifen (T), norgestrel (Ng), norethisterone (Nt) and medroxyprogesterone acetate (Mp) administration. The units of enzyme activity (cofactors generated or utilised) are as described in Table 2 and are presented in comparison with values obtained from uteri of random cycling animals (C). The values are the mean f SD of three independent determinations. Statistical analysis was by Student’s r-test (one-tailed); significantly greater than androgenised: (a) P < 0.025; (b) P < 0.01; (c) P < 0.005; (d) P < 0.0025. creased following progesterone treatment (Table 3) although when expressed relative to DNA however remained unchanged. This suggests that the effect on specific activity was a reflection of an increase in mitochondrial protein. There was no significant change in MDH activity following progesterone treatment whether expressed relative to protein or DNA.

DISCUSSION

The attenuated response of the uterus of the androgenized rat to oestrogenic stimulation [5-81 despite elevated tissue glucose and glycogen suggests an uncoupling of metabolic potential and tissue function. The observed decrease in the activities of PFK and G-6-PD, respectively, key enzymes of the glycolytic and pentose phosphate pathways for the intermediary metabolism of glucose, would support this hypothesis. In the normal uterus PFK [13] and G-6PD [14] synthesis is induced by oestradiol. However, administration of oestradiol at 24 h intervals for greater than 72 h results in the uterus becoming refractory to stimulation and the synthesis of G-6-PD is decreased [15]. Such a refractory state induced in the uterus of the androgenized rat exposed to constant oestrogenic stimulation may therefore explain the apparent decrease in enzyme activities of the glycolytic and pentose phosphate pathways. In liver and uterus a change in glycolysis is accompanied by parallel qualitative changes in the activities of PFK and PK [16, 171. It was surprising therefore

that changes in PFK in the uterus of androgenized rats were not accompanied by changes in PK. Further, PFK activity was increased following each progestin treatment (Fig. 1) whereas the activity of PK remained unchanged (data not shown). The recent description of hormone sensitive low and high molecular weight regulators of PFK [ 18, 191 which do not affect PK may explain these observations in the androgenized uterus. In addition to regulating pentose synthesis, G-6PD activity generates reducing equivalents in the form of NADPH particularly important in lipid biosynthesis (201. Similarly the activities of cytosolic NADP-linked isocitrate and malic dehydrogenases are important sources of reducing equivalents for biosynthetic processes being part of a group of functionally related enzymes involved in the pyruvate/malate cycle. The decrease in activity of each of these enzymes therefore provides further evidence of decreased metabolic activity in the androgenized uterus. The activity of ICDH (NADP), carbon precursors for lipid which supplies synthesis [21] was increased following each progestin treatment in comparison with the limited response of GdPD and MDH. The relative contribution of each of the pathways of the intermediary metabolism of glucose to lipogenesis has been demonstrated to be tissue and hormone specific [22-241. Thus in the uterus of the androgenized rat the stimulation of ICDH activity in response to progestins may provide the precursors and reducing equivalents for lipogenesis associated with the hormone induced differentiation [9, 10, 251.

Uterine enzyme activities

The consistent increase in PFK and ICDH (NADP) following progesterone, norgestrel, norethisterone and medroxyprogesterone acetate, but not tamoxifen, suggest these effects to be related to the progestogenic rather than antioestrogenic properties of these compounds. The regulation of uterine PFK during implantation in the rat is thought to result from selective gene expression in response to appropriate differentiation signals [17,26]. The increase of PFK and ICDH in the present study may therefore represent a biochemical response to a differentiation signal to which the uterus of the androgenized rat has not previously been exposed. Progesterone receptors which potentially could mediate the effects of natural progesterone and synthetic progestins are present in the uterus of the androgenized rat [6], albeit in diminished amounts. Most mitochondrial proteins are encoded by nuclear genes, translated on cytoplasmic ribosomes and imported into mitochondria [27]. Electrochemical gradients and a limited number of receptor and transport pathways are thought to be important mechanisms that regulate protein import and mitochondrial biogenesis [28,29]. The hormonal regulation of these processes is suggested by the decrease in mitochondrial protein and its increase subsequent to progesterone treatment in the uteri of androgenized rats. The maintenance of ICDH activity relative to DNA despite a 3-4fold decrease in mitochondrial protein, suggests that the synthesis and/or turnover of this enzyme is protected in the androgenized uterus and would explain the increase in its apparent specific activity. A similar preferential effect on MDH would explain the increase in its specific activity despite a decrease relative to DNA in the androgenized uterus. The cellular response to oestradiol is mediated, at least in part, by specific receptors for this hormone. A diminution in uterine sensitivity to oestradiol occurs during ageing that has been associated with changes in the capacity of the oestrogen receptor to interact with cell nuclei [30,31]. Attenuation of the induction by oestradiol of PFK [32], GdPD and progesterone receptor [33] has also been described in the ageing uterus and suggested to result from physicochemical changes in oestrogen receptor and its nuclear interaction [33]. Our previous observation of the failure of oestradiol to induce progesterone receptor [6] together with the decrease in PFK and GdPD reported herein would suggest some analogy with the attenuated response to oestradiol observed during ageing. We are currently investigating gene expression in the uterus of androgenized and normal rats to further define the cellular basis of the attenuated response to oestradiol.

REFERENCES

1. Barraclough C. A. and Fajer A. B.: Modification in the CNS regulation of reproduction after exposure of pre-

495

pubertal rats to steroid hormones. Recent Prog. Horm. Res. 22 (1966) 503-539. 2. Gorski R. A.: Gonadal hormones and the perinatal development of neuroendocrine function. Frontiers in Neuroendocrinology

(Edited by L. Martini and W. F. Ganong). Oxford University Press, New York (1971)

pp. 237-290. 3. Cheng H. C. and Johnson D. C.: Serum estrogens and gonadotropins in developing androgenized and normal female rats. Neuroendocrinology 13 (1973) 357-365. 4. Barraclough C. A. and Fajer A. B.: Progestin secretion by gonadotropin-induced corpora lutea in ovaries of androgen-sterilized rats. Proc. Sot. exe. Biol. Med. 128 (1968)781-785. 5. Lob1 R. T.: Androgenization: Alterations in the mechanism of oestrogen action. J. Endocr. 66 (1975) 79-84. 6. White J. O., Moore P. A., Elder M. G. and Lim’ L.: The relationship of the oestrogen and progestin receptors in the abnormal uterus of the adult anovulatory rat. Biochem. J. 1% (1981) 557-565. 7. Wrenn T. R., Wood J. R. and Bitman J.: Oestrogen responses of rats neonatally sterilized with steroids. J. Endocr. 45 (1969) 4!5420.

8. Lob1 R. T. and Maenza R. M.: Androgenization: Alterations in uterine growth and moroholoev. -, Biol. Reprod. 13 (1975) 255-268.

9. White J. O., Moore P. A., Elder M. G. and Lim L.: Progesterone therapy results in partial reversability of uterine abnormalities of the adult anovulatory rat. Biochem. J. 202 (1982a) 535-541. 10. White J. O., Moore P. A., Marr W., Elder, M. G. and Lim L.: Comparative effects of progesterone, norgestrel, norethisterone and tamoxifen on the abnormal uterus of the anovulatory rat. Biochem. J. 208 (1982b) 199-204. 11. Burton K.: A study of the conditions ‘and mechanisms of the diphenylamine reaction for the colorimetic estimation of deoxyribonucleic acid. Biochem. J. 62 (1956) 315-323.

12. Lowry 0. H., Rosebrough N. J., Farr A. L. and Randall R. J.: Protein measurement with Folin phenol reagent. J. biol. Chem. 193 (1951) 265-275. 13. Singhal R. L., Valdares J. R. E. and Ling G. M.: Metabolic control mechanisms in mammalian systems 1. Hormonal induction of phosphofructokinase in the rat uterus. J. biol. Chem. 2b2 (1967) 2593-2598. 14. Barker K. L.. Adams D. J. and Donohue J. R. T. M.: Regulation of the levels of mRNA for glucose-6phosphate dehydrogenase and its rate of translation in the uterus by estradiol. In Cellular and Molecular Aspects of Implantation (Edited by S. R. Glasser and D. W. Bullock). Plenum Press, New York (1981) pp. 269-278. 15. Smith E. R. and Barker K. L.: Effects of estradiol and nicotinamide adenine dinucleotide phosphate on the rate of synthesis of uterine glucose-&-phosphate dehydrogenase. J. biol. Chem. 249 (1974) 65416547. 16. Weber G., Singhal R. L., Stamm N.‘B., Lea M. A. and Fisher E. A.: Synchronous behaviour pattern of key glycolytic enzymes. In Advances in Enzyme Regulation. (Edited by G. Weber). Pergamon Press, Oxford, Vol. 4 (1966) pp. 59-81. 17. Surani M. A. H. and Heald P. J.: Changes in enzymes of carbohydrate metabolism in rat uterus during early pregnancy. Acta endocr., Copenh. 68 (1971) 815-816. 18. Van Schaftingen E., Hue L. and Hers H. G.: Control of the fructose-6-phosphate/fructose 1,6-biphosphate cycle . . . _. m isolated hepatocytes by glucose and glucagon. LIIOthem. J. 192 (1980) 887-895. 19. Kruep D. A. and Dunaway G. A.: Properties of the phosphofructokinase regulatory factors. Archs Biothem. Biophys. 235 (1984) 512-520. 20. Fritz I. B.: Factors influencing the rates of long-chain

fatty acid oxidation and synthesis in mammalian systems. Physiol. Rev. 41 (1961) 52-129.

496

Z. ScRw~arz

et al.

21. D’Adamo A. F. and Haft D. E.: An alternate pathway

28. Schatz G. and Butow R. A.: How are

Chem. 240 (1965) 613-617. 22. Brown J., McLean P. and Greenbaum A. L.: Influence of thvroxine and luteinizina hormone on some enzymes concerned with lipogenesisin adipose tissue, testis and adrenal gland. Biochem. J. 101 (1966) 197-203. 23. Aruldhas M. M., Valivullah H. H. and Govindarajulu P.: Effect of Thyroidectomy on testicular enzymes of the pyruvate/malate cycle involved in lipogenesis. Biochim. biophys. ACM 755 (1983) 90-94. 24. Yoshimoto K., Makanura T. and Ichihara A.: Reciprocal effects of epidermal growth factor on key lipogenic enzymes in primary culture of adult rat hepatocytes. J. biol. Chem. 258 (1983) 1235512360. 25. Warren J. C. and Crist R. D.: Effects of ovarian steroids on uterine metabolism. In Handbook of Physiology (Edited by R. 0. Greep and E. B. Astwood). American Physiological Society, Washington, D.C. Section 7, Vol. Vol. II, Part 2 (1973) pp. 49-68. 26. Heald P. J.: Biochemical Aspects of Implantation. J. Reprod. Ferr. Suppl 25 (1976) 29-52. 27. Schatz G.: How mitochondria import proteins from the cytoplasm. Febs. Len. 103 (1983) 201-211.

29. Mori M., Matsue H., Miura S., Tatibana M. and Hashimoto T.: Transport of proteins into mitochondrial matrix. Eur. J. Biochem. 149 (1985) 181-186. 30. Haii M.. Chuknviska R. S. and Roth G. S.: Isolated uterine nuclei and cytosol receptors of aged rats exhibit impaired estrogenic-stimulation of RNA-polymerase II. Proc. natn. Acad. Sci. U.S.A. 81 (1984) 7481-7484.

31. Chuknyiska R. S., Haji M., Foote R. S. and Roth G. S.: Age-associated changes in nuclear binding of rat uterine estradiol receptor complexes. Endocrinology 116 (1985) 537-551. 32. Singhal R. L., Valadares J. R. E. and Ling G. M.: Estrogenic regulation of uterine carbohydrate metabolism during senescence. Am. J. Physioi. 217 (1969) 793-797.

33. Belisle S., Bellabarba D., Lehoux J-G., Robe1 P. and Baulieu E. E.: Effect of aging on the dissociation kinetics and estradiol receptor nuclear interactions in mouse uteri: Correlation with biological effects. Endocrinology 118 (1986) 75&758.