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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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A Stephanou, L Myatt, A L W Eis, N Sarlis, H Jikihara and S Handwerger

Abstract

During human placental differentiation, mononuclear cytotrophoblast cells fuse and differentiate into syncytiotrophoblast cells. Although syncytiotrophoblast cells have been shown to express interleukin-1α (IL-1α), IL-1β and IL-6, the pattern of expression of these cytokines during placental differentiation is unknown. We have examined the expression of IL-1α, IL-1β and IL-6 mRNA during differentiation of cytotrophoblast cells in culture. IL-1α, IL-1β and IL-6 mRNA levels were determined by semiquantitative reverse transcription-PCR analysis using glyceraldehyde phosphate dehydrogenase as an internal control. All three cytokine mRNA levels decreased markedly during trophoblast differentiation. After 6 days in culture, when almost all the cytotrophoblast cells had fused and differentiated into syncytiotrophoblast cells, the amounts of IL-1α, IL-1β and IL-6 mRNA were decreased by 87·1, 72·1 and 60·9% respectively. Exogenous IL-6 had differential effects on cytokine mRNA expression. When added to placental cultures during the first 6 days of culture, IL-6 markedly inhibited IL-6, IL-1α and IL-1β mRNA expression. However, when added to the cells during days 6–9 of culture, when most of the cells were syncytiotrophoblast cells, IL-6 stimulated IL-lα and IL-1β mRNA expression. The results of these studies indicate that IL-1α, IL-1β and IL-6 mRNA expression decreases markedly during cytotrophoblast differentiation in vitro and that the regulation of trophoblast cytokine mRNA levels changes during differentiation.

Journal of Endocrinology (1995) 147, 487–496