Oestrogen receptors in the genital tract of the Australian marsupial Trichosurus vulpecula

GENERAL

AND

COMPARATIVE

Oestrogen

Receptors in the Genital Marsupial Trichosurus C. E.

Department

46, 417-427 (1982)

ENDOCRINOLOGY

of Physiology.

YOUNG Monash

AND

I. R.

Utziversity,

Tract of the Australian Vulpecula MCDONALD Clayton,

Victoria

3168, Australia

Accepted March 16, 1981 Macromolecules with high binding affinity for oestrogens (K, = ItIs-10’” Mm’ at 0”) are detected in cytosols of vaginal and uterine endothelium of the marsupial brush-tailed possum Trichosurus vulpecula. There are two distinct classes, of high (8 S) and low (4 S) molecular weight, the proportions of which vary in relation to the reproductive state or hormone treatment. In the endometrium, the 8-S form predominates during all phases of the breeding cycle: but there is a relative increase in the 4-S form, up to 40% of the total, during lactation. In the vagina1 endothelium, an 8-S form is predominant in cytosols from follicular and lactating animals, with little or no 4-S binding, but the reverse occurs in luteal or pregnant animals. Only the 4-S form is found in cytosols of endometrium or vaginal endothelium of 16-day castrate females. After treatment of castrates with oestradiol, testosterone, or progesterone, however, only the 8-S form is detected in the endometrium. In the vaginal endothelium, the high-affinity binding remains in the 4-S region in castrates treated with oestradiol or testosterone, but is in the 8-S form after treatment with progesterone. The concentration of high-affinity binding sites per milligram of protein is significantly increased in both endometrial and vaginal endothelial cytosols during the follicular phase of the cycle and in castrates treated with oestradiol. This increase is associated with a marked increase in size of the two organs. Treatment with progesterone or testosterone has no effect on the concentration of binding sites in castrates. These findings are discussed in relation to the unique method of reproduction in marsupials.

The most complete information on the broad sequence of anatomical and histological changes in the ovaries and genital tract of a marsupial is that of the seasonally breeding brush-tailed possum Trichosurus vulpecda (Pilton and Sharman, 19621. The female brush-tailed possum is monovular and polyoestrous with an oestrous cycle of about 26 days in length when isolated from the male. During the oestrous cycle progressive changes occur in the size and weight of the uteri and medial vaginae. The lateral vaginae, which are much smaller structures, exhibit changes similar to those of the medial vaginae. The changes correlate with variations in ovarian activity, so that a short 5- to 7-day follicular phase, characterized by maturation and ovulation of one follicle, is accompanied by a marked increase in size of the vaginal complex and a lesser increase in size of the

uteri (Plate 1). In the uteri there is evidence of epithelial proliferation and in the vaginae cornification of the epithelium. These changes are associated with an increase in the ovarian secretion of “oestrogenlike” substances (Thorburn et al., 1971). Ovulation is followed by luteinization of the ruptured follicle; this is associated with further increase in size of the uteri but regression of the vaginal complex. Histologically the endometrium enters a secretory phase which persists until Day 12 after ovulation and then wanes, with a decrease in uterine weight and necrosis of the superficial epithelial cells with replacement from below. These events are accompanied by a rapid rise in plasma-progesterone concentration beginning about 4 to 5 days after ovulation, reaching a maximum at Days 12 to 14, and then declining rapidly back to the preluteal level (Thorburn et nl., 1971: 417 0016.6480/82/040417-11$01.00/O Copyright All rights

Q 1982 by Academic Press, Inc. of reproduction in any form reserved.

418

YOUNG AND MC DONALD

PLATE 1. Photographs of the reproductive tracts of female possums. Upper: (L) lactating, (A) anoestrous, and (F) follicular states. The size of the bladder (Bl) provides a reference for the size of the reproductive tract in the several states. UGC, urogenital canal: MV, medial vagina: LV, lateral vagina; U, uterus. Lower: (T) 16-day castrate animals treated with testosterone, (P) progesterone, and (E) oestradiol. Note the similarities between the androgen-treated and lactating, progesterone-treated and anoestrous, oestrogen-treated and follicular animals.

Shorey and Hughes, 1973). The supervention of pregnancy does not influence these endocrine events for that cycle and an immature young is born about 18 days after ovulation at the end of the “oestrous cycle.” Thereafter the joey is suckled in the pouch for about 5 months, oestrous being inhibited during this lactation. This is associated with regression of the ovaries and

a reduction in size of the genital tract as occurs during seasonal anoestrous. It is generally accepted that, in eutherian mammals and avian species, a cytoplasmic oestrogen-binding protein in oestrogensensitive cells interacts with the oestrogen to form a complex which translocates to the nucleus, where it initiates gene expression in the form of cytoplasmic protein synthesis

MARSUPIAL

OESTROGEN

and a physiological response (Glascock and Hoekstra, 1959; Jensen and Jacobson, 1962; Gorski, 1973). This investigation was made to determine whether or not the sequential changes in the reproductive tract of the female marsupial could be correlated with similar oestrogen binding. METHODS Animals. The reproductive condition of mature female possums (1.8-3.4 kg body wt) was assessed by histological examination of the ovaries and the presence or absence of a suckling joey. Reproductive status was classified as anoestrous, lactating, or oestrous. The oestrous animals were further subdivided as follicular, luteal, or pregnant. Treatment. One group of 39 females was gonadectomized to reduce endogenous oestrogen levels and sacrificed 3 days later by ether overdose. A second group of 20 gonadectomized possums either was given no further treatment or was injected (im) once with 0.2 mgikg oestradiol valerate (Primogyn, Schering), daily with 0.01 mg/kg oestradiol-3-benzoate (Sigma) for 13 days, once with 3 mg/kg testosterone oenanthate (Primoteston, Schering), or daily with 2 mg/kg progesterone for 13 days (Proluton, Schering). These animals were sacrificed 16 days after commencement of treatment. The 3-day interval between castration or cessation of treatment was selected to reduce the effect of translocation of steroid-receptor complex to the nucleus on cytosol receptor content and it is known to stabilize the cytoplasmic receptor level in the rat (Cidloweski and Muldoon, 1974; Dionne et al., 1980). Tissue cytosol preparation. Animals were anaesthetized with ether and blood samples were taken by cardiac puncture within 15 min of induction. They were then killed by anaesthetic overdose. The uteri and medial vaginae were rapidly excised and placed in ice-cold 0.05 M buffer, pH 7.4 (0.05 M Tris-HCl, 2 m&4 EDTA, and 2 mM mercaptoethanol, termed buffer A). The tissues were rinsed in buffer several times, blotted on filter paper, and weighed. All the remaining procedures were carried out in a cold room at 0 to 4”, unless otherwise specified. The uteri and medial vaginae were split longitudinally and the endothelia were scraped off with a scalpel blade and collected in appropriate volumes of buffer A. Each tissue from individual animals was homogenized separately, in glass-Teflon homogenizers immersed in an ice bath, care being taken to avoid undue heating. The paste was then centrifuged at 105,OOOg in an MSE 65 Mk 2, Superspeed ultracentrifuge for 1 hr at 4”. The clear supematant fraction was decanted and immediately subjected to the various analytical procedures. Blood samples were allowed to clot for 1 hr at room

RECEPTORS

419

temperature and then maintained at 0 to 4” overnight. The serum exudate either was used immediately or stored at -18” for later use. Cytosol incubations. Purified [3,4,6,7-3H]oestradiol-17P (specific activity 85 Wmmol, Amersham) in ethanol was added to polystyrene tubes and evaporated to dryness; serum or cytosol fractions were added to give a final concentration of 0.4 to 0.8 r&f. For competition studies increasing amounts of unlabelled steroids dissolved in ethanol were added with the radioactive steroid prior to evaporation and addition of the cytosol. Receptor assay. Quantification of the high-affinity binding parameters in tissue cytosols was achieved by the gel filtration method of Ginsburg et al. (1974). Aliquots of cytosol incubations were chromatographed on small glass columns of Sephadex LH-20 (i.d. = 0.45 mm, I = 60 mm). The free fraction was discarded and the bound fraction was collected in a scintillation vial for measurement of radioactivity. The data from these experiments were plotted according to the method of Scatchard (1949) and the binding curve was subsequently resolved into binding components by the method of Rosenthal (1967). The concentration of binding sites was standardized according to the total cytosol-protein concentration, which was assayed by the method of Lowry et a/. (1951). Binding specificity. The ability of a 40-fold molar excess of unlabelled oestradiol, oestrone, oestriol, progesterone, testosterone, Sa-dihydrotestosterone, and cortisol (Sigma) to compete with the labelled oestradiol for binding to the uterine and vaginal endothelial cytosols, as well as to serum, was assessed by the more convenient dextran-coated (DCC) method developed by Korenman (1968). An ahquot (0.5 ml) of a DCC solution consisting of 1.25% Norit A-activated charcoal (Sigma) and 0.125% dextran T70 (Pharmacia) in 0.05 M buffer A was added to the cytosol or serum incubate and allowed to stand for 15 min. The mixture was then centrifuged for 15 min at 2000g and the bound fraction in the supernatant was retained for measurement of radioactivity. Ultracentrijirgation. Prior to ultracentrifugation, free steroid was removed from the incubates by a modification of the DCC method. The DCC solution was spun at 2OOOg for 15 min. The resulting supematant was discarded and the cytosol or serum incubate was added to the charcoal pellet, mixed, and allowed to stand for 15 min. The solution was centrifuged for 15 min at 2000g and the supernatant was kept for analysis. Aliquots (0.3 ml) of tissue cytosol or serum incubate (with a total protein concentration of less than 2 mgiml) were layered onto 5-ml linear (5-20% (w/v) sucrose gradients in 0.05 M buffer A. Cytochrome c (Sigma), bovine serum albumin (BSA) (Commonwealth Serum Lab., Melbourne, Australia) and beef

YOUNG AND MC DONALD

420

liver catalase (Sigma) were used as standard markers. The gradients were centrifuged in a Beckman L265B ultracentrifuge at 131,OOOg for 17.5 hr, using a SW50 1 rotor. At the end of the run, the gradients containing isotopically labelled steroids were collected in IO- or 20-drop fractions in scintillation vials for determination of radioactivity. The sedimentation coefticients of the cytosol-binding proteins were determined by the method of Martin and Ames (1961). Quantification of radioactivity. The radioactivity from sucrose gradient fractions was measured in 10 ml of a standard toluene scintillator (4 g POP, 0.5 g POPOP, and 1000 ml toluene) with an ISOCAPI300 liquid scintillation spectrometer (Searle Analytic), equipped with 133Ba external standardization for determination of counting efficiency. All large aqueous samples were dissolved in appropriate amounts of a commercial aqueous scintillator (PCS, Amersham-Searle). An LKB-Wallac 8100 liquid scintillation spectrometer, with 26sRa external standardization for quench correction, was used for measurement of radioactivity. Presentation of data. All of the data were presented as a mean (x) + the standard error of the mean (SE). Differences between statistics were deemed significant if the F(ANOVA) or “t” ratio had a probability of occurrence of less than 0.05% (P < 0.05).

RESULTS

Binding Affinity for Oestradiol in Cytosols of the Endometrium and Vaginal Endothelium High-affinity, limited-capacity binding for oestradial was demonstrable in cytosols of both the endometrium and vaginal endothelium. It was found that this binding was near-maximal at 4” 6 hr after incubation commenced and was stable for at least 66 hr after this (Fig. 1). Scatchard plot analyses

; 00 m

0

24 INCUBATION

46 TIME

72 (hr )

FIG. 1. Time-course of [3H]oestradiol-17p binding, displaceable by a lOO-fold molor excess of oestradiol17p, in cytosol from the median vagina of T. ~ulpeculu.

of the binding data indicated the presence of two classes of binding sites, one representing a high-affinity, limited-capacity and the other a low-affinity, high-capacity site (Figs. 2a and b). No relationship between the reproductive state and the high-affinity association constant was apparent (Table I), and so the data from each tissue cytosol from all females were combined for statistical purposes. The mean association constants for the uterine and medial vaginal binding were 2.7 + 1.3 (SE) x 1O’O and 7.3 “_ 1.0 x lo9 M-l, respectively. The corresponding values for the high-capacity system in the uterus and medial vagina were 3.4 ? 1.0 (SE) and 2.3 + 0.8 x 107M-I, respectively. The binding-affinity constants of cytosols from either the endometrium or the vaginal epithelium were not altered by treatment with progesterone or oestradiol benzoate for 16 days (Table 1). Similarly, administration of testosterone oenanthate had no effect on the affinity constants of the vaginal endothelial cytosols. The endometrium of animals treated with testosterone oenanthate was sparse and there was insufficient material for quantitation of the binding parameters. Concentration of Limited Capacity Binding Sites in Cytosols of the Endometrium and Vaginal Endothelium A distinct relationship between the reproductive state of the female possum and the concentration of high-affinity oestradiol binding sites in cytosols of the endometrium and vaginal endothelium was found (Table 1). The relative concentrations in endometrium were: lactating = anoestrous < luteal (P < 0.05) = follicular. There was a similar hierarchy for the vaginal endothelium: lactating = anoestrous < luteal (P < 0.05) < follicular (P < 0.05). In females treated with oestradiol valerate, the concentrations of oestradiol binding sites in endometrial and vaginal endothelial cytosol were within the range of val-

MARSUPIAL

2.5

OESTROGEN

421

RECEPTORS

A

2.0

OESTRADIOL

-

x 10gM

2. Scatchard plots of oestradiol-17/I binding to cytosol from the (0) media1 vagina endothelium and (0) endometrium. The curves were resolved into high (K ha. -) and low (K,,, -) afftnity binding components according to the method of Rosenthal (1967). The total protein concentration was 3.3 mgi ml in the vaginal cytosol and 2.0 mg/ml in the uterine cytosol. (A) K,, = 1.4 X 10” M-r, K,, = 1.O X 10” M-‘; (B) Kha = 4.0 x 10”’ M-‘, K,, h 3.7 x lo7 M-'. FIG.

TABLE

1

THE HIGH-AFFINITY ASSOCIATION CONSTANT (K,,) AND THE CONCENTRATION FOR OESTRADIOL-170 IN CYTOSOLS OF THE ENDOMETRIUM AND VAGINAL

Endometrium

Group Mature females Follicular Luteal Lactating Anoestrous Females treated with steroids Oestradiol valerate Testosterone oenanthate Progesterone Control

KhZA (10-L” x M-1)

OF BINDING SITES (n) ENINTHELIUM

Vagina1 endothelium

n(10” X moV mg cytosol protein) 0.54 0.36 0.14 0.12

1.3 rc_0.46 (4)

0.75 k 0.31

0.44 2 0.15 (4)

0.42 + 0.22

0.34 t 0.06 (5) 0.46 k 0.09 (4) 0.58 k 0.10 (3)

0.13 t 0.04

1.18 k 0.07

k 0.48

(4)

0.28 0.10 0.03 0.04

1.7 2 0.88 (4) 2.1 c 1.1 (3)

0.14 + 0.05

0.87 0.71 0.70 0.69

a 0.30 (4) 4 0.26 (7)

n(10r2 x mot/ mg cytosol protein)

0.91 k 0.41 (9) 6.1 -c 4.4 (7) 2.3 IC 1.6 (6)

1.2

t k 2 t

Kh, (lo-‘0 x M-1)

k 0.10 (9)

+ 0.18 (7)

1.29 0.44 0.24 0.15

0.12 0.12

t 0.38 ‘- 0.10

2 0.07 2 0.06

” 0.02 f 0.03

Note. One group of animals was either castrated for 16 days (control) or castrated and treated with oestradiol valerate, testosterone oenanthate, or progesterone for 16 days. The reproductive status of a second group of animals was determined and they were then castrated 3 days before the tissues were obtained. Values represent the mean + SE with the number of animals in parentheses.

422

YOUNG

AND

ues found in oestrous animals and greater than those of control animals (P -=c0.05). In contrast, the concentration of high-affinity oestradiol binding sites in cytosols of vaginal endothelium from females treated with testosterone or progesterone were similar to those of control animals, as was the concentration of endometrial binding sites in animals treated with progesterone. Binding

Specijkity

The specificity of high-affinity oestradiol binding in cytosols of the endometrium and vaginal endothelium was determined from the evidence of competition for binding between [3H]oestradiol and a 40-fold molar excess of either oestradiol, oestrone, oestriol, progesterone, testosterone, Sa-dihydrotestosterone (SCPDHT), or cortisol. Because there was no detectable influence of reproductive state on the specificity of binding to the oestradiol binding component, the data from all females were combined for statistical purposes. The reduction in bound counts caused by addition of nonradioactive oestradiol was arbitrarily designated as 100% displacement from limited-capacity binding sites. The reduction in counts caused by addition of an excess of the other unlabelled steroids could then be expressed as a percentage of the reduction due to oestradiol displacement. Results from these competition experiments are presented in Table 2. In both tissue cytosols the only significant displacement, relative to oestradiol, was by the other two oestrogens tested, oestrone and oestriol. These compounds displaced 35-55% of the counts displaced by oestradiol in cytosols of uterus or medial vagina, and there was no significant difference between the two tissues. Ultracentrifugation Tissue cytosols were centrifuged on a linear 5-20% (w/v) sucrose gradient in buffer A to determine the Svedberg (S)

MC

DONALD

coefficients of the oestradiol-binding moieties. The results from these experiments are presented in Table 3. Endometrial cytosols from follicular, luteal, lactating, and pregnant females contained a limited-capacity oestradiol binding component with a sedimentation coefficient of 8 S. Specific binding in the 4-S region was minimal (
MARSUPIAL

OESTROGEN

RECEPTORS

TABLE INHIBITION

COMPETING

UNLABELLED Reduction

Unlabelled competitor

100 35

Oestriol Testosterone

55 3.1

Sa-DHT Progesterone Cortisol is the mean oestradiol

IN THE

PRESENCE

in limited-capacity

[3H]oestradiol

binding

Vaginal 100 39

k8

k5 ir 2.3

52 4.8

18 k 3.5

results from designated

of cytosols from females treated with oestradiol valerate revealed a diffuse band of limited-capacity binding, extending through the 0- to 4-S region of the gradient. The possibility that the limited-capacity binding might be due to contamination of the cytosols with serum proteins was investigated by incubating samples of possum serum with [3H]oestradiol. Ultracentrifugation revealed the presence of a 4-S component, but addition of a IOO-fold molar excess of oestradiol did not reduce this binding. DISCUSSION

The tindings demonstrate that cytosols of the endometrium and vaginal endothelium from the marsupial T. vufpecula contain macromolecular steroid hormone binding components which are of limited capacity, specific for oestrogens, and have a high affinity for oestradiol with association constants of approximately log to lOlo M-l, at 0”. The inability to detect similar oestrogen binding components in the plasma or sera from these animals suggests that these entities are localized within the tissue cell, although the possibility that they are situated in the extracellular compartment cannot be excluded. These components fulfill some of the criteria currently used to define a steroid hormone receptor and can be considered as such.

6.1 0.46 0 individual as 100%

animals displacement

2 SE. from

(%)

endothelium

-c9

1.9 +- 3.2 5.0 k 2.7 0 of at least six was arbitrarily

OF

STEROIDS”

Endometrium

Oestradiolb Oestrone

0 Each value * Nonradioactive

2 BINDING

OF [3H]OEST~~IOL-17/!l

423

k5.6 k 3.0

limited-capacity

binding

sites.

The physical properties of the cytosol oestrogen receptors in the endometrium and vaginal endothelium, as characterized by ultracentrifugation and association constant for oestradiol, are similar to those described for eutherians (see King and MainWaring, 1974, for review). An anomaly exists, however, between the two groups in their relative binding affinity for naturally occurring oestrogens. SEDIMENTATION

TABLE 3 COEFFICIENTS

OF LIMITED-

CAPACITY OESTRADIOL BINDING IN CYTOSOLS OF ENDOMETRKIM VAGINAL ENDOTHELIUM

Castrate females treated with steroids Oestradiol valerate Oestradiol benzoate Progesterone Testosterone oenanthate Controls

Vaginal endothelium

Endometrium

GKClp Mature females Follicular Luteal Pregnant Lactating

COMPONENTS AND

8 8” 8” 8”

4”

(2) (1) (2) (2)

8” 8”

8”

4”

(I)

-

8” 8”

4” 4”

(1) (2)

8

4 -

8”

4”

(2)

-

4

(2)

-

4

(2)

-

4

(2)

Note. Values were taken to the nearest whole the number of animals are shown in parentheses. ’ Major species present. * Minor species present.

4” 4 4 4”

<4

(2) (1) (2) (2)

(1) (I) (2)

number:

424

YOUNG AND MC DONALD

BSA

CATALASE

J

170

BSA

CATALASE

J

J

J

0I

150

1

J MEDIAL

4 R

-1 0

5

10

15 20 GRADIENT

O TOP

30

FRACTION

FIG. 3. Sedimentation profiles of cytosols of uterus and vaginal endothelium from (A) a 16-day castrate and (B) a lactating female. Cytosols were incubated with (0) [3H]oestradiol or (0) [3H]oestradiol plus 100-fold molar excess of unlabelled oestradiol. BSA. bovine serum albumin.

In Tvichosurus the endometrial and vaginal endothelial cytosol oestrogen receptor had a higher affinity for oestriol than for oestrone; although, in the endometrium, this was only significant at the P < 0.01 level. In many eutherian species, however, oestrone typically is bound preferentially over oestriol in the reproductive tract (Korenman, 1969; Elsner et al., 1977; Saiduddin and Zassenhaus, 1977). Oestriol is produced by the fetoplacental unit in relatively large quantities during pregnancy in several eutherian species (Jirku and Layne, 1964; Beling, 1971) and is effective in the stimulation of early uterotrophic events such as water imbibition, RNA and protein synthesis (Anderson el al., 1972; 1975), although its precise function at this time has not been fully delineated. It is tempting to

speculate that the relative enhancement of oestriol binding in Trichosurus, as compared to eutherian species, may be related to the minor degree of placentation and abbreviated pregnancy that is characteristic of marsupials generally. The physicochemical characteristics of the oestrogen receptors in the endometrial cytosol did not change significantly during the reproductive cycle, whereas those of the vaginal endothelium underwent profound changes. The high-molecular-weight (8 S) form predominated during the follicular, lactating, and anoestrous phases of the breeding cycle, while only a low-molecular-weight form (4 S) could be detected during the luteal phase in both pregnant and nonpregnant animals. This finding suggests that agents released during the luteai

MARSUPIAL

OESTROGEN

RECEPTORS

425

absence of the 8-S receptor in the endometrium of the castrate animal indicates 8000 that the nature or concentration of hormones present under these circumstances MEDIAL is incompatible with production of this reVAGINA ceptor form. The apparent absence of the 8-S receptor from the vaginal cytosol of oestrogentreated animals may have been due to preferential saturation of the 8-S receptor binding sites by oestrogen and subsequent movement of the receptor complex into the nuclei. This would imply a significant $ structural and functional difference beMEDIAL tween the 4- and 8-S receptor forms. VAGINA Treatment of animals with oestradiol valerate at a dose of 200 pg/kg also apparently produced a variety of low-molecular-weight binding components up to that of the 4-S I 1 I species. The reason for this unusual disperTOP 5 10 15 sion of the receptor material is unclear. It GRADIENT FRACTION may reflect pathological breakdown of the FIG. 4. Sedimentation profiles of [YH]oestradiol-17P binding in cytosols of medial vagina from (A) a luteal receptor as a consequence of overdosage or and (B) a follicular female (0) with and (0) without it may simply be a technical artifact due, for addition of lOO-fold molar excess of oestradiol-176 example, to the induction of nonspecific proteases (Notides et al., 1973, 1976). It did phase may convert an 8-S receptor to a not, however, occur during any stage of the 4-S one, this process being reversed on natural breeding cycle or in the gonadecwithdrawal of these agents. Progesterone is tomized animal and is presumably not of an obvious candidate (Hsueh, Peck, and physiological relevance. Clarke, 1976), as it is present in maximal Long-term treatment of possums with concentrations during the luteal phase of the oestradiol, but not testosterone or progesincreased the recepoestrous cycle (Shorey and Hughes, 1973). terone, markedly This explanation seems unlikely, however, tor:protein ratio in both the endometrium and vaginal endothelium as compared to as treatment of the castrate animals with progesterone produced predominantly an 16-day castrate animals. The magnitude of 8-S form, in direct contrast to the effect of this effect may have been underestimated due to occupation of the binding sites by progesterone in the rhesus monkey (Elsner exogenous oestrogen and translocation of ef ul., 1977). It is apparent that the characteristics of the complexes to the nuclei. Therefore it the oestrogen receptor in the endometrium seems probable that changing levels of endiffer from those in the vaginal endothedogenous oestrogen may regulate cytosol hum. Only in the endometrium was indivioestrogen-receptor levels in the vaginal endual administration of either oestrogen, dothelium and endometrium and be at least progesterone, or testosterone able to pro- partly responsible for the differences in duce mainly the 8-S form. This is consistent oestrogen binding capacity observed during with the predominance of the 8-S species the reproductive cycle. Long-term oestrothroughout the entire breeding cycle. The gen administration also has been shown to BSA

CATALASE

426

YOUNGANDMCDONALD

augment the levels of cytoplasmic oestrogen receptor in oestrogen-responsive tissues of eutherian species (Pavlik and Coulson, 1976). A similar mechanism, if operant in Trichosurus, may contribute to the lower level of receptor in vaginal endothelium during the luteal phase, as compared to the follicular phase, in response to increasing levels of plasma progesterone (Shorey and Hughes, 1973). The possibility that the observed receptor forms are artifacts, produced during homogenization of the tissue, must be mentioned. The inability to mimic in vivo conditions, in terms of protein concentration and ionic strength as well as various other factors, also indicates the need for caution in extrapolation to the physiological situation. Furthermore, the 3-day period after castration or cessation of treatment which elapsed before the tissues were sampled, may not have been ideal for stabilization of the receptor in this species. Despite this, the consistently tissue-specific nature of oestrogen binding observed in the vagina and endometrium, as well as the changes related to the breeding cycle or after hormone administration, suggests that the physiochemical characteristics of the receptors in these tissue are distinctly different. The preparative conditions were identical in all cases. Whether this difference resides in the molecular structure of the receptor or is a consequence of other tissuespecific entities, such as ionic content or proteolytic enzyme activity, is unknown. The structural differences, however, could be part of a process that enables these tissues to respond differentially to the same hormonal milieu and could also be an efficient means of modulating the interaction of oestrogen with the cellular constituents, thereby inducing changes in cellular function. In summary, it is clear that either the follicular state or oestrogen administration is associated with a significant increase in the concentration of macromolecular high-

affinity oestrogen binding sites in both the endometrium and the vaginal endothelium of these marsupials, the effect being greatest in the vaginal endothelium. Furthermore, the characteristics of these macromolecules in the two organs are affected differently by the reproductive state or by reproductive hormone administration. ACKNOWLEDGMENTS This research was supported by Grant Dl 75116070 from the Australian Research Grants Committee and a Commonwealth Graduate Scholarship awarded to C.E.Y.

REFERENCES Anderson, J. N., Clark, J. H., and Peck, E. J., Jr. (1972). The relationship between nuclear receptor, estrogen binding and uterotrophic responses. Biochem. Biophys. Res. Comrnun. 48, 14601468. Anderson, J. N., Peck, E. J., and Clark, J. H. (1975). Estrogen-induced uterine responses and growth: Relationship to receptor estrogen binding by uterine nuclei. Endocrinology 96, 160-167. Beling, C. G. (1971). In “Endocrinology of Pregnancy” (F. Tucks and A. Klapper, eds.), p. 32. Harper & Row, New York. Cidlowski, J. A., and Muldoon, T. G. (1974). Estrogenic regulation of cytoplasmic receptor populations in estrogen-responsive tissues of the rat. Endocrinology 95, 1621- 1629. Dionne, F. T., Dube, J. V., Frenette, G., and Tremblay, R. R. (1980). Effects of endocrine manipulations on oestrogen binding in cytosols from rat skeletal and perineal muscles. J. Endocrinol. 85, 351-358. Elsner, C. W., Blingworth, D. V., de Groot, K., Fleckinger, G. L., and Mikhail, G. (1977). Cytosol and nuclear estrogen receptor in the genital tract of the rhesus monkey. J. Steroid Biochem. 8, 151- 156. Ginsburg, M., Greenstein, B. D., Maclusky, N. M., Morris, D., and Thomas, P. J. (1974). An improved method for the study of high-affinity steroid banding: Oestradiol binding in brain and pituitary. Steroids 23, 773-792. Glascock, R. F., and Hoekstra, W. G. (1959). Selective accumulation of tritum-labelled hexoestral by the reproductive organs of immature female goats and sheep. Biochem. J. 72, 673-682. Gorski, J. (1973). Estrogen binding and control of gene expression in the uterus. In “Handbook of Physiology,” Section 7: Endocrinology, Vol. 2, pp. 525-536. Williams & Wilkins, Baltimore. Hsueh, A. J., Peck, E. J., Jr., and Clark, J. H. (1976).

MARSUPIAL

OESTROGEN

Control of uterine estrogen receptor levels by progesterone. Endocrino/og.v 98, 438-444. Jensen, E. V., and Jacobson, H. I. (1962). Basic guides to the mechanism of estrogen action. Recent Prog. Horm. Res. 18, 387-414. Jirku, H., and Layne, D. S. (1965). The metabolism of oestrone-14C in a pregnant chimpanzee. Steroids 5, 37-44. King, R. J. B., and Mainwaring, W. I. P. (1974). “Steroid-Cell Interactions,” Chapters 3,4,7, and 9. Univ. Park Press, Baltimore. Korenman, S. G. (1968). Radio ligand binding assay of specific estrogens using a soluble uterine macromolecule. J. Clin. EndocrinoL 28, 127-130. Korenman, S. G. (1969). Comparative binding affinity ofestrogens and its relation to estrogenic potency. Steroids

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