Effect of cortisol on estradiol secretion and its reception in the morning and evening hours in 30-day-old female rats

Cortisol administration (250 micrograms) to 5-day-old female rats was studied for its effect on the diurnal (morning-evening) rhythm of the estradiol secretion and reception on the 30th day of life. This stimulus had no influence on the estradiol level in the morning time. In the evening the estrogen concentration in the control group decreased while in the cortisol-treated group there were no changes. Disturbance of the circadian periodicity in the serum estradiol concentration in cortisol-treated animals was not induced by changes in the secretory ability of adrenals and ovaries, which was estimated by measurement of the estradiol production by glands in vitro. This disturbance was not a result of changes in hormone reception in uterine cytosol. The increased estradiol level in cortisol-treated rats in the evening is in good agreement with effects caused by neonatal glucocorticoid treatment in the reproductive system.     

Effect of malignant tumors with various characteristics on the state of the estradiol receptor system in peripheral target organs

Experiments on 24 randombred rats with growing lymphosarcoma and mammary carcinoma have demonstrated alterations in steroid-producing function of the ovaries and adrenals and in the estradiol-receptor system. These alterations were found to be inconclusive and dependent on the characteristics of the growing tumor. In rats bearing lymphosarcoma, the reduced estradiol levels in the blood and ovaries were in agreement with a negligible number of cytoplasmic and nuclear receptors in the uterus, mammary glands and tumor. Rats with mammary carcinoma demonstrated an increase in estradiol levels in the blood, ovaries and adrenals and a dramatic fall in the blood progesterone level. In this case the number of cytoplasmic receptors in the uterus and mammary glands decreased, whereas that in the nucleus dramatically rose. The measurement of the number of estradiol molecules in the nucleus and cytoplasm and its correlation to the receptor number made it possible to reveal substantial differences between the estradiol-receptor systems in the two tumors regardless of the low level of receptors in both tumors.     

Immunocytochemical localization and enzymatic distribution of rat ovarian aromatase

A rabbit antiserum directed against purified human placental aromatase was used for immunohistochemical localization of the enzyme in rat ovaries. Immunostaining was conducted on tissue from animals at various ages and in different reproductive states: immature; immature, eCG-treated; immature pseudopregnant; adult cycling; and adult pregnant. Various labeling protocols were employed (e.g. horseradish peroxidase-conjugated secondary antibody, peroxidase-antiperoxidase, and avidin-biotin-peroxidase on fresh frozen and Bouin's fixed paraffin-embedded sections), but the avidin-biotin-peroxidase method on paraffin sections proved to be superior to the others. In immature rats, most of the immunostaining, which was quite weak, was limited to the stroma. After stimulation with eCG, some of the granulosa cells of antral follicles exhibited immunostaining; in pseudopregnant rats, most staining occurred in the luteal cells. In mature animals, the corpora lutea of pregnant and cycling rats demonstrated the greatest degree of immunostaining. No significant immunoreactivity was detected in pre-antral follicles, but in early antral follicles and preovulatory follicles, both theca and granulosa cells exhibited immunostaining. Aromatase enzymatic activity was also determined on ovarian microsomal fractions of eCG-treated immature animals, pregnant animals at term, and cycling animals. Furthermore, enzyme activity and estradiol concentrations were examined after ovaries from proestrous rats were dissected into follicular, luteal, and residual components. Activity was found in all regions and correlated with immunostaining and estrogen production. These results argue against a model in which all the immunoreactive/enzymatically active protein is localized in granulosa cells of Graafian follicles and suggest that corpora lutea may be involved in estrogen synthesis during the rat estrous cycle as well as during pregnancy.     

Hormone secretion by euthyroid and hypothyroid rat ovaries during the early stages of hCG-induced ovarian cyst development

This study was undertaken to examine ovarian steroid production during the early stages of hCG-induced ovarian cyst formation in the hypothyroid rat. Rats were placed into two groups with one group made hypothyroid by adding thiouracil to their diet. After 10 days, each group was divided into two subgroups with one subgroup receiving daily injections of hCG for 2 days and the other subgroup receiving saline. On the morning of Day 13, ovaries were removed and incubated for 2 hr. No significant difference in progesterone secretion was observed. However, ovaries from hypothyroid, hCG-treated rats secreted significantly more testosterone and estradiol than ovaries from vehicle-treated, hypothyroid rats and euthyroid, hCG-treated rats. In a second experiment, ovaries from euthyroid and hypothyroid rats treated with hCG were incubated in medium supplemented with 100 nM androstenedione and 0 or 100 ng FSH/ml. FSH failed to affect progesterone, testosterone, and estradiol secretions by ovaries from euthyroid, hCG-treated rats. In contrast, FSH significantly enhanced testosterone and estradiol secretion by ovaries from hypothyroid, hCG-treated rats. These results support the hypothesis that increased levels of testosterone and estradiol secretion have a central role in the induction of polycystic ovaries by hCG in the hypothyroid rat.     

In vivo evaluation of 7 alpha-[11-(4-[125I]iodophenoxy)undecyl]-17 beta-estradiol: a potential vector for therapy of adrenal and estrogen receptor-positive cancers

7 alpha-[11-(4-[125I]Iodophenoxy)undecyl]-17 beta-estradiol ([125I]IPUE2) was synthesized and its tissue distribution studied in immature female Fischer rats. Upon intravenous administration, [125I]IPUE2 was shown to accumulate in the adrenals and, to some extent, in the uterus and the ovaries. Coinjection with estrogen receptor (ER)-saturating quantities of unlabeled 17 beta-estradiol did not significantly reduce the uptake of [125I]IPUE2 in these tissues. The high adrenal uptake of [125I]IPUE2 is most likely associated with the lipophilic nature of the 7 alpha-substituted estradiol. The potential to use the 7 alpha-undecylestradiol as a vector to direct therapeutic groups to adrenal and ER-positive cancers is discussed.     

Immunological demonstration of alpha-fetoprotein in uterine cytosol from immature rats

An immunological demonstration of alpha-fetoprotein (AFP) in uterine cytosol from immature rats is reported. The identity between serum AFP and the estrogen binding 4.5 S macromolecular complex of uterine cytosol was demonstrated by the use of a specific immunoadsorbent to AFP. Analysis of the sedimentation profile in glycerol gradients of uterine cytosol incubated with tritiated estrone or estradiol suggests that the total estrogen binding capacity of the 4.5 S complex is provided by AFP. Changes of AFP content in rat uterus with the age of the animals suggest that this protein is probably present in the cytosol as a serum contaminant.     

Modifications in adrenal activity during hepatocarcinogenesis by N-2-fluorenylacetamide

During chemical hepatocarcinogenesis by N-2-fluorenylacetamide, the hormonal status of female Sprague Dawley rats is largely modified. Adrenals present a modified activity which is evidenced by an enlargement of glomerulosa and reticularis zones and a decrease of the fasciculata, while medulla remain unchanged. The level of aldosterone remain quite normal in N-2-fluorenylacetamide treated rats despite changes observed in the glomerulosa. The cortisol level was lowered in agreement with the decreased size of the fasciculata. The estradiol level was normal despite the reduced activity of the ovaries. The possibility of a participation of the reticularis zone in the regulation of the estradiol level is suggested. The possible protective action of the adrenals during hepatocarcinogenesis is discussed.     

Heparin-releasable lipase activity of rat adrenals, ovaries and testes.

The presence of NaCl-resistant, neutral triacylglycerol hydrolase (lipase) activity in rat adrenal gland, ovary and testis was studied. Both adrenals and ovaries but not testes were found to contain such a lipase. The activity of the enzyme in the adrenal gland was lowered during cortisol treatment and hypothyroidism. An elevated adrenal lipase activity was found during hyperthyroidism. Pseudo-pregnant and lactating rats had higher ovarian lipase activities than cyclic rats. Ovarian lipase activity in lactating rats was positively correlated with the serum concentrations of progesterone and of 20 alpha-hydroxyprogesterone and negatively correlated with the high-density-lipoprotein non-esterified cholesterol concentration. The lipase activity of adrenals and of ovaries was largely releasable from these organs by heparin and could be inhibited by an antibody against heparin-releasable liver lipase. This indicated that the lipase is extracellularly located and is similar to 'liver' lipase. A possible role of this lipase in adrenals and ovaries is discussed.     

Estradiol secretion by the ovary of 19-day-old hypophysectomized and sham-operated chick embryos

The amounts of estradiol released into culture media by ovaries from 19-day-old hypophysectomized (decapitated) and sham-operated chick embryos were determined by RIA. Per single ovary, ovaries from decapitated embryos secreted slightly less estradiol than ovaries from sham-operated ones during a 4-h culture period (837 +/- 413 vs 935 +/- 378 pg), but this difference was not significant. On an ovarian weight basis, ovaries from decapitated embryos secreted slightly more estradiol than ovaries from sham-operated ones (1.15 vs 0.91 ng), but this difference was not significant either. It is concluded from these results that the hypophysis does not control estradiol secretion by the ovary in the 19-day-old chick embryo.     

Ovarian contents of immunoreactive beta-endorphin and alpha-N-acetylated opioid peptides in rats

beta-Endorphin was measured by radioimmunoassay in homogenates of ovaries from immature Sprague-Dawley rats (21-29 days of age) and found to be present at levels of about 0.6-0.7 ng/ovary. After administration of PMSG there was approximately a 4-fold increase (2-3 ng/ovary) in total ovarian immunoreactive (ir) beta-endorphin 48 h after injection. Analysis of follicular fluid from similarly treated rats indicated about the same amount of ovarian ir-beta-endorphin (2-3 ng/ovary) as in ovarian homogenates, suggesting that most of the ir-beta-endorphin is localized in follicular fluid of PMSG-primed immature rats. Immature rats were made pseudopregnant by administration of hCG 48 h after PMSG, and at 24 h after injection of hCG there was a slight, but significant and reproducible, increase in the ovarian content of ir-beta-endorphin. The serum concentration of ir-beta-endorphin was in the range of 1-3 ng/ml and was unaffected by PMSG and PMSG/hCG; likewise, the pituitary content of ir-beta-endorphin did not change following administration of gonadotrophins to immature rats. In mature cyclic animals, levels of 2-4 ng ir-beta-endorphin/ovary were found, comparable to those in the ovaries of PMSG-primed immature rats, and there were only small changes during the oestrous cycle. In addition to ir-beta-endorphin, we also obtained evidence for the presence of alpha-N-acetylated opioid peptides (endorphins or enkephalins) in the ovaries of PMSG-primed immature and mature rats. The physiological role of the opioid peptides in reproductive tissue is unknown, but they are presumably acting in an autocrine or paracrine fashion.     

Physiological peripubertal activation of the ovary is not reproduced by pregnant mare serum gonadotropin (PMSG) administration

During the days preceding the first ovulation the ovary of the rat exhibits a remarkable increase in estradiol (E2) and progesterone (P) release in response to gonadotropins. No such increase is observed in the case of androgens (A, testosterone + dihydrotestosterone). The present experiments were undertaken to examine the possibility of reproducing these developmental events by stimulating the ovary with a gonadotropin that has substantial FSH-like activity. In vivo administration of pregnant mare serum gonadotropin (PMSG) to juvenile 29-day-old rats greatly increased the in vitro E2 and A response to human chorionic gonadotropin (hCG) measured 2 days later in the morning. The magnitude of the A response was significantly larger than that of ovaries from juvenile animals or rats in first proestrus. The E2 response was much greater than that of juvenile ovaries but similar to that of ovaries from late proestrous rats. In contrast, the P response to hCG was not enhanced by PMSG. In fact the response was similar to that of juvenile ovaries and markedly less than that of first proestrous rats. This decreased P response was not due to a greater conversion of P to its less active metabolite 20 alpha-hydroxy-4-pregnen-3-one (20 alpha-OH-P). The results suggest that PMSG enhances the E2 and A response of immature ovaries to hCG at the expense of that of P. Treatment of immature rats with PMSG may represent a useful model to study E2 release from preovulatory ovaries, but it cannot be used to reproduce in its entirety the developmental changes in steroidal response to gonadotropins associated with normal puberty.     

Comparative analysis of the interaction of various estrogens with the estrogen-receptor system of the uterus

Equilibrium binding of labeled estron, estradiol, estriol and DES was studied in uterine cytosol of immature Wistar rats. The dissociation kinetics of the ligand complexes with specific high affinity sites suggested the homogeneity of estrogen receptors in rat uterine cytosol. The feasibility of intracellular regulation of estrogen action in target cells both at the receptor and post-receptor levels is discussed.     

Estradiol-17β receptors in the immature rat ovary

Estradiol binding components in the cytosol and nuclear fractions of the ovary from immature rats (22–28 days old) were characterized by $\text{in vitro}$ methods. Several of the biochemical characteristics of the estradiol binding components in the ovarian tissue were compared with the estradiol receptor from the uterus. The results suggest that the ovarian estradiol binding components are similar to the specific high affinity estradiol receptors in the uterus. In the cytosol of intact rat ovary a significant fraction of the total binding sites was found to be occupied, presumably by the endogenous estrogen. Following hypophysectomy there was a significant increase in the available cytosol binding sites. Evidence for translocation of cytosol receptor-estrogen (RE) complex to the nucleus was obtained for the ovary. The sedimentation properties of the RE complex of the ovary and the uterus are similar. The ovarian cytosol RE complex sediments at 7-8S in glycerol gradients at low ionic strength and at 4S in sucrose gradients at high ionic strength. Following extraction with 0.4 M KCl the ovarain nuclear RE complex sediments at 5S in sucrose gradients which is identical to that of the uterine nuclear receptor.     

Synthesis and secretion of an estrus stage-specific protein by rat uterus.

A specific protein with an estimated molecular weight of 260 kDa was found to be synthesized and secreted into the incubation medium by rat uterus only during the estrus stage of the cycle. This secreted uterine protein was designated as estrus stage-specific protein (ESP). ESP was not produced by pregnant, lactating or immature pup rat uteri. Estradiol administered to ovariectomized rats induced production of ESP which was blocked by the antiestrogen, ICI 182, 780. The present results show that the synthesis and secretion of ESP is regulated by estradiol and this protein maybe involved in blastocyst implantation.     

Androgen receptor in rat liver: Hormonal and developmental regulation of the cytoplasmic receptor and its correlation with the androgen-dependent synthesis of α2u-globulin

The cytoplasmic receptor for 5α-dihydrotestosterone has been identified in the rat liver and partially characterized. The receptor is a protein with a sedimentation coefficient of 3.5 S and binds both androgens (5α-dihydrotestosterone and testosterone) and estradiol-17β with high affinity. At saturating concentration, for every mole of estradiol there seem to be three moles of 5α-dihydrotestosterone bound to the receptor. Whereas estradiol stronly inhibits the uptake of 5α-dihydrotestosterone by the receptor, the presence of 5α-dihydrotestosterone only weakly interferes with estradiol binding.The level of the androgen receptor activity in the hepatic cytosol was found to follow closely the level of the urinary output of α-_2u-globulin, an androgen-dependent protein of hepatic origin. Immature and senile male as well as female rats, which do not normally produce α_2u-globulin, also lacked androgen receptor activity in their hepatic cytosol. Castration of the adult male rats results in a gradual drop of the urinary output of α_2u-globulin as well as of the hepatic androgen receptor activity. Androgen treatment of immature and senile male rats does not induce α_2u-globulin or any receptor activity. Administration of estradiol to adult male rates results in complete inhibition of both α_2u-synthesis as well as complete loss of the cytosol androgen receptor activity in these animals. These results strongly indicate that the hepatic the hepatic androgen receptor activity. Androgen treatment of immature and senile male rats does not induce α_2u-globulin or any receptor activity. Administration of estradiol to adult male rats results in complete inhibition of boty α_2u-synthesis as well as complete loss of the cytosol androgen receptor activity in these animals. These results strongly indicate that the hepatic androgen receptor is an inducible protein whose synthesis is regulated by its own ligands, the androgens acting as the positive and the estradiol as the negative signals.     

Effects of estrogen and testosterone on specific prolactin binding in the kidneys and adrenal of rats.

The effects of estradiol benzoate in the female rat, testosterone propionate in the male rat, and castration in both sexes on specific prolactin binding sites in the particulate membranes of the kidneys and adrenals were studied. Castration resulted in a significant increase in PRL binding activity in the kidneys of both males and females, and in a significant increase in PRL binding activity in the adrenals of the females. The increase in PRL binding with castration and the decrease seen with testosterone treatment were similar in both immature and mature rats. Progesterone administration to castrate females failed to alter PRL binding in both tissues. The present results suggest that estrogen and testosterone participate in the PRL osmoregulatory system in rat.     

Binding of high-density lipoproteins to luteal membranes: the role of prolactin, luteinizing hormone, and circulating lipoproteins

Ovarian and adrenal membranes from immature gonadotropin-primed rats, treated with 4-amino-pyrazolopyrimidine (4APP) to reduce endogenous lipoprotein levels, displayed higher binding of porcine high-density lipoprotein (HDL) when compared to control rats. Immature, hypophysectomized (HYPOX) rats bearing corpora lutea (CL) on Day 5 after ovulation had lower levels of serum progesterone and reduced capacity for HDL and human chorionic gonadotropin (hCG) binding to ovarian membranes when compared with intact animals. Hypophysectomy also reduced the number of HDL binding sites in adrenal membranes. Treatment of HYPOX animals with luteinizing hormone (LH) and prolactin (Prl) alone or in combination increased the HDL binding sites in the ovary relative to HYPOX-untreated rats. Neither hormone affected binding to adrenals, where only adrenocorticotropic hormone (ACTH) enhanced HDL binding. LH treatment reduced the serum progesterone levels and hCG binding to the ovaries, whereas Prl administration increased progesterone levels with no effect on hCG binding. We conclude from this study that HDL binding in the luteinized ovary is regulated by Prl and LH and circulating lipoproteins, whereas in adrenals it is regulated by ACTH and circulating levels of lipoproteins.     

A 17-kDa protein induced in rat myometrium by hormonal treatment

A 17-kDa protein mainly recovered in the myometrium fraction sedimenting at 10,000 g has been induced by estradiol treatment in spayed rats. Tamoxifen completely inhibited the effect of estradiol, and progesterone decreased it by 50%. The protein was scarce in unspayed animals and the level after estradiol treatment remained much lower than in spayed rats. Oral doses of the potent antiprogesterone RU 38486 increased the level of the 17-kDa protein in unspayed animals. It was not present in immature or gestating rats or in the endometrium or myometrial cell cytosol of any of the rats. Following electrophoretic purification, it bound calmodulin, but was not phosphorylated by cAMP protein kinases.     

Follicle-stimulating hormone plays a role in the induction of ovarian follicular cysts in hypophysectomized rats.

Unabated stimulation by low doses of LH-like activity produces ovarian follicular cysts in both progesterone-synchronized immature rats and pregnant rats. Serum FSH is maintained in both of these models at values similar to those observed on diestrus. To determine whether unabated stimulation by basal serum FSH affects the ability of LH-like activity to induce cystic ovaries, immature hypophysectomized (HYPOXD) rats were given either no hormone (control); 2 micrograms ovine FSH (oFSH) once daily for 14 days beginning on Day 27; 0.5 IU hCG twice daily for 13 days beginning on Day 28 of age; or both oFSH and hCG (FSH + hCG) beginning on Day 27 and Day 28, respectively. By the end of the in vivo treatments (Day 40 of age), the largest follicles in the ovaries of control and hCG-treated HYPOXD rats were at the preantral stage of development, whereas the largest follicles present in ovaries from FSH-treated animals were atretic and at the small antral stage of development. In contrast, ovaries from rats treated with FSH + hCG displayed large follicular cysts by Day 37 of age. Of the serum steroids analyzed, only estradiol and androstenedione concentrations for animals treated with FSH + hCG were consistently elevated above values observed for control HYPOXD rats. Serum testosterone and dihydrotestosterone values were similar for hCG-treated and control HYPOXD rats throughout the in vivo treatments. In contrast, these steroids were elevated between Days 3 and 5 of FSH treatment (+/- hCG treatment). Serum progesterone and estrone values for all in vivo gonadotropin treatment groups were similar to those of controls. Serum insulin concentrations were not affected by any in vivo treatment. Incubates of follicles/cysts from FSH + hCG-treated HYPOXD rats contained more progesterone, androstenedione, and estradiol than incubates of follicles from any other in vivo treatment group. Follicles from all in vivo treatment groups responded to 8-bromo cAMP (cAMP) with increased in vitro progesterone accumulation. However, only follicles from FSH-treated and FSH + hCG-treated rats responded to cAMP with increased androstenedione and estradiol accumulation in vitro. Inclusion of 400 ng of either androstenedione or testosterone in the incubation medium enhanced progesterone accumulation in follicular incubates from control, hCG-treated, and FSH-treated HYPOXD rats, but did not enhance progesterone accumulation in follicular incubates from FSH + hCG-treated animals. Both androstenedione and estradiol production increased markedly under these conditions for follicles from all in vivo treatment groups.(ABSTRACT TRUNCATED AT 400 WORDS)