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G. B. Kudolo, M. G. Elder and L. Myatt

ABSTRACT

Numbers of granulosa cells obtained from follicles of immature rats increased from 1·6 × 105 cells/ovary on day 8 to 7·1 × 106 cells/ovary on day 40 of age, the day of vaginal opening and first pro-oestrus. Very high levels of cytosol oestrogen receptor were found on day 8 (175 000 sites/cell) but by day 19 20 000 sites/cell were found. Nuclear receptor concentrations were highest on day 12(5400 ± 1470 (s.d.) sites/cell) and again on day 21 (5400 ± 2300 sites/cell). After day 21 both cytosol and nuclear oestrogen receptor concentrations fell and remained low until nuclear concentrations rose at day 40. Two consecutive daily injections of FSH/LH (5 i.u.) increased cell number over control in animals killed on day 22, gave no significant alteration in animals killed on day 26 or 28 but decreased numbers in animals aged 32 and 35 days. Only on day 22 was the increase in cell number associated with an increase in nuclear oestrogen receptor concentrations. Indeed on days 32 and 35 increased nuclear receptor concentrations were associated with a decreased cell number.

J. Endocr. (1987) 112, 333–338

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G. B. Kudolo, M. G. Elder and L. Myatt

ABSTRACT

Rat granulosa cell cytosol contains a second oestrogen-binding species (SOB) distinguished from the classical oestrogen receptor by its lower dissociation constant (approx. 45 nmol/l) and the ability to bind oestrogens, antioestrogens, androgens and progesterone but not diethylstilboestrol. The SOB and the oestrogen receptor can be further distinguished by their differential adsorption to spheroidal hydroxylapatite and Concanavalin A–Sepharose. Addition of chaotropic salts or molybdate to granulosa cell cytosol did not alter the concentration of SOB or oestrogen receptor measured, indicating that there are no 'masked' binding sites in the two species caused by aggregation phenomena. The association rate of oestradiol with SOB at 4°C (1·72 ± 0·27(s.e.m.) × 108 mol/h) and 25°C (4·50 ± 0·36 × 108 mol/h) was faster than with the oestrogen receptor (7·20 ± 0·15 × 107 mol/h and 1·23 ± 0·15 × 108 mol/h respectively). The biphasic dissociation kinetics of [3H]oestradiol from the oestrogen receptor at 25°C (rate constants k −1 = 0·30±0·07/min and k −2 = 3·73±0·57 × 10−3/min) were similar to those reported in other target tissues but the dissociation of [3H]oestradiol from SOB appeared to be much more rapid and could not be measured by the Sephadex LH-20 separation method employed for determining receptor kinetics. Using sucrose density-gradient (SDG) analysis and Sephacryl S-200 gel chromatography the oestrogen receptor fractionated in an aggregated form (10·3S, Stokes radius >5·2 nm) in low ionic strength buffers and as a small species (4·4S, Stokes radius 3·5 nm) in buffers containing 0·4 m-KCl. However, the SOB fractionated as 2–3S, Stokes radius 3·7–4·0 nm at low ionic strength and as 5·8S, Stokes radius 3·5 nm in 0·4 m-KCl. In contrast to the receptor from other target tissues the granulosa cell oestrogen receptor did not bind to the artificial acceptor matrix oligo(dT)-cellulose and heat activation did not promote a 4S to 5S conversion when analysed on SDG. The salt-extracted form of nuclear receptor sedimented at 4·6S, mol. wt 69–72 000 on SDG.

J. Endocr. (1984) 102, 93–102

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G. B. Kudolo, M. G. Elder and L. Myatt

ABSTRACT

The dissociation constants (K d) and steroid specificities of oestrogen-binding species in rat granulosa cell cytosol and nuclei have been studied. Preliminary work, where diethylstilboestrol was employed as competitor in binding assays, identified the oestrogen receptor in whole ovarian tissue nuclei (K d 0·35 ±0.09 nmol/l) and cytosol (K d 0·39 ± 0·03 nmol/l). Isolation of granulosa cells revealed that the majority of this receptor (75–96%) was present in these cells. Specificity studies on the binding of [3H]oestradiol in granulosa cell cytosol indicated the presence of an additional class of oestrogen-binding sites which were, however, not present in nuclei. Saturation analysis over an extended range of [3H]oestradiol concentrations and using unlabelled oestradiol as competitor revealed a binding species of K d 45·8± 6·9 nmol/l (capacity 16·7 pmol/mg cytosol protein) for oestradiol in addition to the cytosol oestrogen receptor of K d 0·58 ± nmol/l (capacity 2·8 pmol/mg cytosol protein). The low affinity of this novel species implies that the dextran-coated charcoal techniques used in previous studies on ovarian oestrogen-binding species would cause dissociation of ligand and not allow it to be measured.

The second oestrogen-binding species displayed affinity for oestradiol-17β, oestriol, oestrone, testosterone, 5α-dihydrotestosterone, methyltrienolone, progesterone and the antioestrogens tamoxifen, nafoxidine and clomiphene citrate. The species, however, did not bind diethylstilboestrol, a characteristic shared with other low affinity cytosol oestrogen-binding species which have been reported in dog prostate, chick oviduct and male rat liver but not shared with uterine type II oestrogen receptors. It can be further distinguished from the oestrogen receptor by differential ammonium sulphate precipitation and the stability of its ligand binding at temperatures above 55 °C where the oestrogen receptor–ligand interaction is rapidly lost.

Concentrations of nuclear oestrogen receptor in granulosa cells (2200 sites/cell) were similar to those found in other target tissues but a high proportion of this receptor (70%) was 'unoccupied' or available for binding at 4 °C and the majority (75%) was resistant to extraction with 0·4 m-KCl. As the second oestrogen-binding species could not be detected in granulosa cell nuclei it is unlikely to be involved directly in eliciting genomic responses to hormonal stimulation. It is more probable that it regulates the level of the free intracellular steroid to which the oestrogen receptor of the granulosa cell (the predominant site of oestrogen biosynthesis) is exposed.

J. Endocr. (1984) 102, 83–91

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G. B. Kudolo, F. N. Mbai and R. M. Eley

ABSTRACT

Sixteen individually caged adult female vervet monkeys (Cercopithecus aethiops), whose reproductive parameters had been studied for 5 years, were hysterectomized/ovariectomized during three reproductive states; i.e. the late follicular (15·4 ± 4·7 (s.d.) days) and luteal (27·8 ± 4·7 days) phases of the normal cycle (20–50 days), and during prolonged intermenstrual intervals (PII; 99·0 ± 2·5 days since the previous menses). These latter animals showed characteristics of both follicular and luteal phases; i.e. their ovaries contained both corpora lutea and large antral follicles and systemic oestradiol and progesterone concentrations were raised. Analysis of cytoplasmic oestrogen and progestin receptors (CER and CPR) revealed that endometrium during PII had CER levels of 0·58 ± 0·07 pmol/mg protein, similar to those of the follicular phase (0·60 ± 0·12); CPR levels (1·20 ± 0·87) were not different from those of the luteal phase (1·05 ± 0·45). The ratio of CPR to CER during the luteal phase was about tenfold higher than that of the follicular phase. Levels during PII were intermediate between the two phases. Under receptoractivating conditions, the DNA-binding components of the PII cytoplasmic fraction underwent over 40% loss while those present during both phases of the normal cycle doubled. The hormone-binding sites at all times remained intact indicating that the DNA and hormone-binding sites are distinct on both CER and CPR.

Less than 50% interaction of CER/CPR with DNA-cellulose was obtained, indicating the presence of only limited quantities of cytoplasmic activating factors which may be a prerequisite for receptor binding to the genome. During PII, factors which deactivate DNA-binding sites may also have been induced. Extensive accumulation of nuclear oestrogen receptor was evident in PII endometrium with 80% being salt-resistant. This level is higher than that in the follicular and luteal phases (37 and 52% respectively). These data, suggesting a possible aberration of receptor activation in vitro and receptor processing in vivo, may be indicative of endometrial dysfunction during PII. This could lead to a delay in menstruation.

J. Endocr. (1986) 110, 429–439