E. Ekka

Estrogen-receptors and Estrogen-induced Protein-synthesis in the Uterus of Diabetic Rats

SummaryThe present study was undertaken to examine whether the diabetic state influenced the early stages of oestrogen action in the uterus. Ovariectomized streptozotocin diabetic rats were given intravenous infusions of oestradiol-17 B one day to 4–7 months after the initiation of diabetes. Oestradiol was taken up to a similar extent (on a wet weight basis) by the uteri of diabetic and control animals. Oestradiol receptor levels were similar in the cytosol of control and diabetic rats (1.06±0.19 and 1.01±0.27 pmol/uterus respectively). Nuclear translocation of the receptor complex under the influence of oestradiol infusion occurred similarly in both groups. The stimulation of a specific oestrogeninduced protein in the uteri occurred at a similar rate in both groups from one day to 4–7 months after the onset of diabetes. Thus streptozotocin diabetes of short to long duration does not interfere with the early stages of oestrogen action.

Oestrogen and progestogen receptors in endometrium and myometrium at the time of blastocyst implantation in pregnant diabetic rats.

SummaryA suitable hormonal environment is a prerequisite for blastocyst implantation. Experimental diabetes was previously shown to modify the hormonal milieu and produce alterations in oestrogen receptor kinetics in the uterine tissue. In the present work, oestrogen and progestogen receptor levels were measured on the morning of day 6 of pregnancy in normal and in streptozotocin-induced diabetic rats, both in implantation sites and in interembryonic segments of endometrium and myometrium. Receptor levels were different in the implantation sites compared to the interembryonic segments of endometrium, both in the control and in the diabetic animals. Indeed, implantation sites were characterized by lower oestrogen receptor levels in cytosol and higher progestogen receptor levels in cytosol and nuclei. However, compared to the control rats, the diabetic rats had lower oestrogen receptor levels in implantation sites, both in cytosol and nuclei. In the myometrium, the differences between sites or between types of rats were minimal. Plasma levels of oestradiol were lower in diabetic rats than in control animals, whereas progesterone levels were similar. A 20% lower implantation rate was found in diabetic rats, compared to normal rats. These results show that the specific distribution of oestrogen and progestogen receptors between implantation sites and interembryonic segments was preserved in the diabetic rats; however the absolute level of oestrogen receptor was lower. This abnormal endocrine milieu might arise from a lower oestradiol level and a decreased oestradiol/progesterone ratio in the circulating blood. Whether the lower implantation rate in diabetic rats might be a consequence of the overall disturbed hormonal status remains to be elucidated.

Estradiol-induced progesterone receptor synthesis in normal and diabetic ovariectomized rat uterus

Estrogen stimulation of progesterone-receptor (Prog R.) synthesis is an important parameter of the sex hormones activity at the uterine level. Experimental diabetes in the rat has been shown to perturb protein synthesis in some tissues and to reduce, under certain circumstances, estrogen and androgen activity on their respective target tissues. The present work tended to evaluate the effect of streptozotocin diabetes on estradiol (E2) stimulation of Prog. R and on Prog R. kinetics in the rat uterus. Two groups of diabetic rats were primed for three consecutive days with 5 microg. E2 s.c. (EP). One group received an acute i.p. injection of progesterone (P), 1 h before sacrifice (Inj), the other group did not (n Inj). Two other groups, not primed with E2 (nEP) were similarly injected or not with P. Four groups of non diabetic animals served as controls. Estrogen priming induced a 20-25% increase in DNA content, both in controls and in diabetics. Protein content was also increased to almost the same extent in diabetics and controls; protein concentration remained however slightly lower in cytosol of EP diabetics as compared to controls. Prog R. increased about 7-fold in cytosol and 4-5-fold in nuclei of EP control and diabetic groups. Cytosol to nuclei ratios of Prog R. decreased similarly in Inj. EP diabetics and controls, compared to the corresponding n Inj. groups. It is concluded that estrogen priming stimulated Prog R., total protein and DNA synthesis to the same extent in diabetic as in control rats Prog R. kinetics was unaltered in diabetics. This finding might be relevant to situations like early pregnancy, when Prog R. levels change rapidly and specifically in relation with the time and the site of implantation.

Enhanced metabolism of [2,4,6,7,-3H] estradiol-17β in the diabetic rat

The peripheral metabolism of [3H]estradiol-17 beta (E2(3)H) was studied in ovariectomized, streptozotocin induced diabetic rats, in an attempt to understand the reduced nuclear retention time and the decreased activity on protein synthesis of E2 in diabetic rat uterus as reported recently. E2(3)H was injected intraperitoneally and the kinetics of 3H metabolites were followed in plasma extracts obtained between the time intervals of 10-210 min after injection. Diabetics accumulated about 15 times more ether insoluble metabolites by the tenth minute as compared to the controls. The metabolites contained relatively more glycurono- and less sulfoconjugates in diabetics. After hydrolysis of either fraction, most of the radioactivity migrated as estrone or was less polar than estrone on LH20 column chromatography. Half of the ether insoluble material was not hydrolyzable by Helix pomatia gastric juice. The ether soluble fraction contained more estrone and non polar material, but less estradiol in the diabetics than in the controls. The plasma disappearance rates of all fractions were faster in the diabetics. For estradiol itself, the half life was two to three times shorter in the diabetics after either intravenous or intraperitoneal injection of the tracer. The metabolic clearance rate of E2(3)H measured by constant intravenous infusion was 1746 +/- 326 ml/h and 1136 +/- 347 ml/h in diabetics and controls (P less than 0.001). In conclusion, E2(3)H was cleared much faster from the peripheral circulation in diabetics, resulting in an early accumulation of ether soluble and non soluble metabolites; the latter contained proportionately less sulfo- and more glycuronoconjugates than in the controls. The rapid disappearance of E2 from the plasma of diabetics may play a role in the shorter retention time of the hormone-receptor complex and the reduced activity of the hormone at the uterine level.

Estrogen Dependent Induction of Low Affinity Binding-sites in the Nuclear Fraction of Rat Uterus

Type II estradiol binding sites characterized by lower affinity and higher capacity than type I receptor sites have been described in rat uterine nuclei. These sites appeared to be dependent on estrogen stimulation. Reducing agents prevented estradiol binding to these sites. In the present study, the situation prevailing in adult rats (Ad) was studied and compared to ovariectomized (Ox) and ovariectomized estrogen prestimulated rats (OxPS). Nuclear precipitate from Ad, Ox and OxPS rats were incubated with tritiated estradiol (E2(3)H) in the presence and in the absence of mercaptoethanol as reducing agent. In the presence of mercaptoethanol, saturation was attained at E2(3)H concentrations above 16 nM. In the absence of reducing agents, a secondary binding was observed in Ad and OxPS which was not saturated at E2(3)H levels up to 80 nM. Non-specific binding obtained with paired aliquots containing 100-fold excess of DES as competitor was not linear but showed a saturation profile, distorting the saturation curve of the specific sites, obtained by subtracting non-specific from total E2(3)H binding. Increasing DES concentrations up to 10,000 nM did not allow to reach complete exchange with E3(3)H ligand bound to specific sites, preventing measurement of binding sites concentration. Incubation of nuclear fractions with increasing concentrations of E2(3)H (up to 6,000 nM) gave a saturation curve with a linear kinetics above 1-2,000 nM, which represented saturation concentration of the specific sites. From this, non-specific and specific moieties could be estimated. Binding capacity of specific sites was of the order of 50-80 pmol uterus. Half saturation was attained between 300 and 600 nM E2(3)H, which approximated the Kdiss of these sites, at variance with the Kdiss of 15-30 nM originally reported for type II binding sites. In conclusion, these results show that secondary binding sites were present in uterine nuclei of Ad and OxPS rats. Binding capacity was about 30-fold higher than that of type I sites. Affinity was however very low, and casts some doubt on the role of these sites as active estradiol binders in physiological situations. Their increase under the influence of estrogen may however be related to some as yet undetermined role.

Non-stoichiometric nuclear-cytoplasmic redistribution of estrogen receptor in adult rat uterus, following estradiol injection.

In immature and ovariectomized rats acutely injected with estradiol (E2), accumulation of estradiol receptor complexes (E2R) from the uterine cytosol to the nucleus has been shown to be quantitative by numerous investigators. In the present study, translocation of E2R from the cytosol to the nuclear fraction in adult and ovariectomized estrogen prestimulated rats was analyzed. Twenty micrograms of E2, dissolved in saline containing 10% ethanol and 1 g% bovine serum albumin (B.S.A.) were injected intraperitoneally to the animals and 2 h later E2R in the cytosol and crude nuclear fractions were assayed by exchange techniques. Unlike a 91% recovery of the depleted cytosol E2R in the nuclear fraction of ovariectomized rats, only 39.2 and 27.5% were recovered in the adult and ovariectomized estrogen prestimulated rat uterus respectively. Moreover, depending on the temperature and duration of nuclear suspension incubation, from 18 up to 80% of the recovered nuclear E2R were solubilized in the incubation medium and nuclear post-incubation washes and could be measured by hydroxylapatite treatment (HAP). Saturation assays showed a plateau from 12 nM E2 3H onwards up to 80 nM. The Kd values computed for the receptors in the nucleus and HAP in all the three groups were of the order of 2 X 10(-9) M. In conclusion, after E2 administration to adult or ovariectomized estrogen prestimulated rats, a stoichiometric recovery of the depleted cytosol E2R in the nuclear fraction was not observed, even when leakage of nuclear receptor into the medium in course of exchange was taken into account. Chronic estrogenization appeared to modify the dynamics of uterine receptor.