Immunohistochemical detection of estrogen receptors in the canine uterus and their relation to sex steroid hormone levels

Cyclic changes in estrogen receptor expression in the uterine tissue of 60 female dogs were evaluated, using an immunohistochemical technique on formalin-fixed paraffin-embedded sections. The expression of estrogen receptors in the uterine horns, body and cervix was quantified by means of an immunohistochemical score. A negative correlation was found between staining scores in the uterine horns and serum progesterone levels. Generally, staining scores in the uterine horns were highest during proestrus, declined during estrus and were lowest during early metestrus. During anestrus high staining scores for estrogen receptors were observed, indicating sensitivity for estrogens in a sexual quiescence stage. Compared with the uterine horns, high staining scores were found in the uterine body and cervix during estrus and metestrus. No positive staining for estrogen receptors was detected in 1 pregnant uterus. Fluctuations in estrogen receptors were more pronounced in endometrial stroma cells than in epithelial cells of the uterine horns. The importance of stromal cells in the sexual cyclicity of the canine uterus should not be underestimated when studying uterine endocrinology and pathology.     

Immunohistochemical detection of androgen receptors in the canine uterus throughout the estrus cycle

Serum androgen levels in the bitch increase during proestrus and remain elevated until metestrus. To find out whether androgens can have a direct impact on the canine uterus, androgen receptors (AR) were identified immunohistochemically in uterine tissue. Androgen receptor distribution in the uterine horns, body and cervix was described during different cycle stages, during pregnancy and in the postpartum period. Nuclear staining for AR was observed in cells of the surface epithelium, glandular ducts, basal glands and stroma of the endometrium, and in myometrial smooth muscle cells. In addition, cytoplasmic staining was observed in epithelial cells from proestrus to early metestrus, when the cells were secretory active, and in stroma cells during pregnancy, suggesting a role for androgens in decidualization. During pregnancy and in the postpartum period nuclear staining for AR was nearly absent. During the estrus cycle stroma cells stained with higher intensities for AR than epithelial cells, supporting the idea that stroma cells mediate some effects of steroid hormones on epithelial cells in the genital tract. In contrast with earlier findings on estrogen receptor-alpha and progesterone receptors, no significant changes in androgen receptor expression were observed during the estrus cycle. Few correlations were found between the staining for AR and serum levels of the sex steroids. The present findings suggest that there is a basal expression of AR in the canine uterus throughout the estrus cycle that may not be influenced by sex steroid hormones.     

The steroid hormone levels and receptors of steroid hormone receptors in rat muscles during physical exertion

It is shown that the testosterone content in skeletal muscles of female albino rats is 2-fold decreased, while the estradiol content-1.5-fold increased and progesteron content showed no changes after systematic physical exercises. The pharmacokinetic investigations showed that androgen half-life in the organism decreased from 8 to 5 h under physical exercises. The amount of androgen receptors in cytosol of skeletal muscles increases from 1.34 +/- 0.08 to 1.71 +/- 0.10 fmol/mg per 1 mg of protein. Kd is 0.40 +/- 0.03 and 0.48 +/- 0.08 nM, respectively. Sensitivity of the organism to hormonal signal play an important role in the metabolism regulation in skeletal muscles along with the hormonal content change during the organism adaptation to physical exercises.     

Changes in the levels of protein and steroid hormones in the plasma and steroid hormone receptors in the uterus of normal cycling guinea pigs

This study deals with the estrous cycle of guinea pigs in relation to sexual behavior, uterine weight, levels of gonadotropins, steroid hormones, and steroid hormone receptors in the uterus. The guinea pigs in this study showed cyclic changes in various reproductive functions broadly similar to other laboratory species studied. The increase in the uterine weight coincided with high concentration of steroid hormones (estradiol and progesterone) secreted during proestrus and estrus. The elevated levels of steroid hormone receptor concentrations in the uterus during these periods also confirm the role of these hormones. The rise in progesterone level from day 14 of the cycle was associated with lordosis and its related behavior. It was noted that the "estrus behavior" is the most accurate external marker for ovulation and sexual receptivity to males. It was also observed that there is an association between follicle-stimulating hormone and luteinizing hormone during the preovulatory period that was not demonstrated in previous studies.     

Changes in sex hormone receptors during administration of progesterone to prevent estrus in the bitch

The development of lesions and the changes in sex hormone receptors were studied in the uteri of bitches under progesterone treatment. Twelve weeks after the onset of treatment, there was atrophy of the endometrium and increased thickness of the myometrium, without cystic dilatation of endometrial glands. This was accompanied by a dramatic reduction in estrogen-alpha and progesterone receptors in all cell types of the uterine wall. By 24 weeks after the onset of treatment, however, the endometrium was thickened due to the development of cysts of endometrial glands, while the myometrium thickness had returned to normal. The estrogen-alpha and progesterone receptors in most cell types of the uterine wall were again within the normal range. These results clarify and reconcile some apparent contradictions in the literature. They show that sex hormone receptors in most cell types of the uterine wall, especially endometrial gland cells and stromal cells, escape progestin (down) regulation after prolonged exogenous administration of progesterone.     

Endotoxin-induced changes in sex steroid hormone levels in male rats

Intravenous administration of Escherichia coli endotoxin (ENDO) was found to induce profound time and dose dependent changes in the serum steroid hormones, oestrone (E1), oestradiol (E2), corticosterone (B), progesterone (P4), 17 alpha-OH progesterone (17 alpha OHP4), and testosterone (T) of intact male rats. These changes were rapid, with a maximal response at 2 h and a return to close to normal values by 4 h. Non-lethal doses (0.01-2 mg/kg) of ENDO induced large increases in oestrogens (3-9-fold), P4 (4-fold) and B (2-3-fold) and decreased serum T (2-fold). The greatest increase in E2 level was seen with an ENDO dose of 2 mg/kg. Serum E1, E2 and T did not change in response to lethal ENDO doses (4-8 mg/kg); B, P4 and 17 alpha OHP4 levels alone were moderately elevated. Systemic mean arterial pressure was unchanged, except at the highest ENDO dose used. Thus, the hormonal responses are unlikely to be the result of hemodynamic changes. Low doses of ENDO did not produce an increase in serum E1 and E2 in adrenalectomized or orchidectomized rats. These results indicate that oestrogens are largely produced in the testis. The aromatization of the testicular and adrenal androgens can be stimulated by glucocorticoid.     

Combined technique of immunohistochemistry and autoradiography for the simultaneous detection of steroid hormone receptors and their ligand

A combined technique of immunohistochemistry and autoradiography was applied to detect progesterone target cells in tissue sections. The radioactively labeled progestin [3H]-Organon 2058 and the progesterone receptor, revealed by antibodies to the receptor molecule, were localized simultaneously in identical cells on the same tissue section. Technical details that make possible combined detection of the nuclear antigen, present in very small amounts, simultaneously with its steroid ligand are described.     

Serum steroid hormone levels in relation to the reproductive cycle ofSebastes taczanowskii andS. schlegeli

Synopsis Changes in serum steroid hormones were studied during the reproductive cycle of two viviparous rockfishes of the genusSebastes. During the annual reproductive cycle of femaleS. taczanowskii, serum levels of estradiol-17β (E₂) gradually increased from their lowest in August to maximal levels towards the end of vitellogenesis in February, and rapidly returned to low levels in pregnant females in April and May. Serum progesterone (prog) fluctuated at low levels throughout the annual reproductive cycle, while 17α, 20β-dihydroxy-4-pregnen-3-one (17α, 20β-diOHprog) levels were high during gestation in May. Serum levels of these steroids were also measured in femaleS. schlegeli at weekly intervals during late vitellogenesis, gestation and post-parturition. In pregnant females, E₂ was high during vitellogenesis, then decreased and remained low throughout gestation. The 17α, 20β-diOHprog levels were low during vitellogenesis, then rapidly increased and remained high during gestation. On the other hand, prog values remained low, with temporal peaks coinciding with the peak of 17α, 20β-diOHprog during gestation. Based on these results and the literature, it is suggested that E₂ is the most important steroid involved in vitellogenesis and that 17α, 20β-diOHprog may play an important role in final oocyte maturation and subsequent gestation.     

Effects of estradiol and progesterone on the reproductive tract and on uterine sex steroid receptors in female lambs

The effects of estradiol-17 beta (E(2)) and progesterone (P) on the reproductive tract and on uterine estrogen receptors and P receptors were studied in 2-mo-old female lambs (n = 11). On Days 0, 1 and 2, E(2) (1 ug/kg, Group E, n = 4), P (0.3 mg/kg, Group P, n = 4) or corn oil (control) vehicle (Group C, n = 3) were administered, and in Day 3 all lambs were slaughtered. Group E (n = 12) had E(2) serum concentrations (mean +/- SEM) of 43.8 +/- 2.2 pmol L , similar to that of the follicular phase; while P concentrations in Group P (n = 12) were similar (2.8 +/- 0.18 nmol L ) to those of the luteal phase of the ewe estrous cycle. The E(2) treatment increased the reproductive tract weight, while P treatment increased only the uterine weight. Both E(2) and P receptors from upper and middle uterine zones (including the myometrium, endometrium and caruncles) were determined by binding assays with tritiated hormones, dextran-charcoal separation and inverse Scatchard analysis. Both the E(2) and P treatments decreased E(2) and P receptor concentrations in upper and middle zones, although the upper zone had higher receptor concentrations than the middle zone (P < 0.01). E(2) receptor concentrations in the upper zone (mean +/- SEM, fmol mg prot) were 1236 +/- 34, 667 +/- 80 and 444 +/- 103 for Groups C, P and E, respectively. The P receptor concentrations were 2434 +/- 135, 1273 +/- 102 and 1536 +/- 213 for the same groups. The high uterine P receptor concentrations allowed P action without prior estrogen priming of female lambs. The present results suggest that E(2) and P might down-regulate their own and each other's receptors during development. The biological responses induced by E(2) and P, as measured by the reproductive tract weight, demonstrated that at an early stage of development uterine receptors are physiologically active.     

Changes in cytosol and nuclear progesterone receptor concentrations in the rabbit uterus and their relation to induction of progesterone-regulated uteroglobin.

To study the relation between steroid receptor concentrations and biological response, we measured cytosol and nuclear progesterone receptors from rabbit uterus under different experimental conditions, and compared receptor values with induction of uteroglobin, a progesterone-regulated protein. A 5-day progesterone treatment (1 mg/kg per day) reduced the nuclear receptor content by 40%, slightly elevated cytosol receptor levels and increased uteroglobin content 3000-fold. Estradiol and tamoxifen altered progesterone-induced changes in the receptor and uteroglobin values: cytosol and nuclear receptors rose significantly, but uteroglobin induction declined markedly, when progesterone treatment was supplemented with estradiol or tamoxifen. A 50% inhibition of progesterone action on uteroglobin synthesis was achieved with 1 μg/kg of estradiol per day. Thus under certain conditions, there is a clear disparity between steroid receptor levels and biological response.     

Characterization of uterine sex steroid receptors in the pig and their variation during the oestrous cycle

The present study establishes and validates an in vitro binding and exchange assay for tissue receptors for oestradiol (E) and progesterone (P) in pig uterus. Both hormones bound to specific cytoplasmic (Rc) and nuclear (Rn) receptor proteins with high affinity. The relative concentrations of the receptors were measured in dissected samples from endometrium and myometrium obtained at late prooestrus, oestrus, and luteal phases of the oestrous cycle. The Scatchard analysis of the oestradiol and R 5020-receptor complex displayed linearity and indicated a single class of high affinity, low capacity binding sites. Significant variations were seen in the binding of E and P to their cytosolic and nuclear receptors, following the changes in the circulating levels of the hormones in blood plasma during the oestrous cycle. Both tissue components, i.e. endometrium and myometrium followed a similar pattern when related to the stage of the oestrous cycle considered. The ERc increased from prooestrus, reaching a maximum at standing oestrus, thereafter decreasing. The concentration of ERn increased from prooestrus towards the early luteal phase, with a significant reduction by day 8 of the cycle. The amounts of PRc were maximal at standing oestrus, remaining high during the early luteal phase, while the PRn showed a linear increase from oestrus onwards throughout the luteal phase.     

Messenger RNA levels of estrogen receptors alpha and beta and progesterone receptors in the cyclic and inseminated/early pregnant sow uterus

The aim of the present study was to investigate differences in the expression of mRNAs for ERalpha, ERbeta and PR in the sow uterus at different stages of the estrous cycle as well as in inseminated sows at estrus and during early pregnancy by use of solution hybridization and in relation to plasma levels of estradiol and progesterone. Uterine samples were collected at different stages of the estrous cycle and after insemination/early pregnancy. In the endometrium, the expression of ERalpha mRNA and PR mRNA was similar for cyclic and early pregnant groups. Both were highest at early diestrus/70 h after ovulation and ERalpha mRNA was lowest at late diestrus/d 19 while PR mRNA was lowest at diestrus and late diestrus/d 11 and d 19. The expression of endometrial ERbeta was constantly low during the estrous cycle but higher expression was found in inseminated/early pregnant sows at estrus and 70 h after ovulation. In the myometrium, high expression of ERalpha mRNA and PR mRNA was observed at proestrus and estrus in cyclic sows and at estrus in newly inseminated sows. Higher expression of myometrial ERbeta mRNA was found in inseminated/early pregnant sows compared with cyclic sows, although significant only at estrus. In conclusion, the expression of mRNAs for ERalpha, ERbeta and PR in the sow uterus differed between endometrium and myometrium as well as with stages of the estrous cycle and early pregnancy. In addition to plasma steroid levels, the differences between cyclic and inseminated/early pregnant sows suggest that other factors, e.g. insemination and/or the presence of embryos, influence the expression of these steroid receptor mRNAs in the sow uterus.     

Intracellular progesterone receptors are differentially regulated by sex steroid hormones in the hypothalamus and the cerebral cortex of the rabbit

The aim of this study was to examine the role of sex steroid hormones in the regulation of intracellular progesterone receptors (PR) in the rabbit central nervous system. We determined PR concentration in cytosol preparations from the hypothalamus, the frontal, tempo-parietal and occipital cortex, by using the specific binding of the synthetic progestin [³H]ORG 2058. PR concentration was higher in the hypothalamus of intact adult females than in that of adult males and prepubertal females, whereas no significant differences were observed in the cerebral cortex of these animals. PR concentration was similar in the three cortical regions analyzed, indicating a homogeneous distribution of PR in the cerebral cortex. The administration of estradiol to ovariectomized animals increased PR concentration in the hypothalamus but not in the cortex. The administration of progesterone to ovariectomized rabbits did not modify PR concentration in any region, however when progesterone was administered after estradiol, it induced a significant diminution in hypothalamic PR concentration without effects in the cortex. These findings suggest that in the rabbit, PR are estrogen regulated in the hypothalamus but not in the cerebral cortex. In the latter, PR are not regulated by progesterone, whereas in the former the estrogen-induced PR are down-regulated by progesterone. Interestingly, hypothalamic PR constitutively expressed in ovariectomized animals are progesterone-insensitive.     

Interaction of antiprogestins with progesterone receptors in rat uterus

Cytosolic and nuclear progesterone receptors (PRc and PRn) under antiprogestin treatment were measured in rat deciduoma and compared with values for contralateral (nondeciduomatous) rat uterine tissue. Uterine PRc and PRn of the progesterone treated group were 101 +/- 8.7 and 4770 +/- 590 fmol/mg DNA respectively. After treatment with antiprogestins STS-557, 5 alpha-DNE, (5 alpha-dihydronorethisterone), 5 alpha-DNG (5 alpha-dihydronorgestrel), RU-22092 and RU-16556, PRc in the nondeciduomatous control horn ranged from 127 to 377 fmol/mg DNA and PRn from 2785 to 17925 fmol/mg DNA. In the decidual tissue, PRc decreased significantly (4.6 +/- 0.8 fmol/mg DNA) on 5 alpha-DNG treatment as compared with the progesterone alone treatment group (147 +/- 3.8). PRn in decidual tissue also decreased maximally on 5 alpha-DNG treatment. These results suggest that the interaction of antiprogestins may not be identical in control uterine tissue and in deciduoma.     

Inhibitory role of sex steroid in the regulation of ovarian follicle-stimulating hormone receptors during pregnancy.

The present study was conducted to determine the role of sex steroids in the regulation of FSH receptors in pregnant rats. In the normal physiological condition, FSH bindings per unit ovarian weight (density of binding) and per 2 ovaries (total binding) increased during days 14-21 gestation. Scatchard plot analyses of the binding suggested that the increase in FSH binding was due to an increase in the number of FSH-binding sites. The plasma FSH concentration in pregnant rats was stable during the receptor change. In contrast, the plasma estradiol-17 beta concentration continuously increased from gestation day 14 to 21, and the testosterone level showed a large peak on gestation day 18. Estradiol-17 beta (one silastic plate containing 13 mg crystal)-implanted pregnant rats during 14-21 days of gestation induced significant decreases in the total FSH binding and ovarian weight on gestation day 21. Estradiol administration increased the plasma estradiol level 2.3-fold but did not change the FSH level. Testosterone or 5 alpha-dihydrotestosterone, a nonaromatizable androgen, did not influence the binding level under the same dose treatment. In contrast, continuous treatment with aminoglutethimide (2 plates containing 20 mg crystal), an inhibitor of adrenocortical steroidogenesis, for 7 days significantly increased the total FSH binding without a significant change in the ovarian weight. The plasma titers of estradiol and testosterone in pregnant rats treated with aminoglutethimide were reduced by 37% and 51%, respectively. Aminoglutethimide did not influence plasma FSH levels. These results suggest that circulating estradiol acts as a negative factor in the regulation of ovarian FSH receptors, at least during the second half of pregnancy. Other factor(s) that is (are) independent of sex steroids and FSH may contribute to FSH receptor induction.     

Interaction of progestins with steroid receptors in human uterus

Norethindrone (17β-hydroxy-19-nor-17α-pregn-4-en-20-yn-3-one) and norethindrone acetate (17β-acetoxy-19-nor-17α-pregn-4-en-20-yn-3-one) interfered to a varying degree, by competitive inhibition, with the binding of progesterone and oestradiol to respective cytoplasmic receptors in the human uterus. Progesterone binding to 4S macromolecule was saturable and co-specific for progestins. Competitors like norgestrel (17β-hydroxy-18-methyl-19-nor-17α-pregn-4-en-20-yn-3-one), 19-norprogesterone, medroxyprogesterone acetate (17α-acetoxy-6α-methylpregn-4-ene-3,20-dione) and compound R₅₀₂₀ (17,21-dimethyl-19-norpregna-4,9-diene-3,20-dione) possessed higher binding affinities for the progestin receptor. The dissociation constant (K_d) for the progesterone–receptor interaction was 0.6–1.6nm and the receptor concentration ranged between 6600 and 8200 sites/cell. Norethindrone and norethindrone acetate competed for the progesterone receptor with inhibition constants (K_i) of 6.8 and 72nm respectively. Gradient displacement and competitive-receptor assays indicated that norethindrone acetate-binding affinity for progestin receptor was approximately one-tenth that of norethindrone and progesterone. The progestins also inhibited oestradiol binding to 4.6S oestrogenic receptor by 8–12%, involving interaction at the oestradiol-binding site with a calculated K_i value of 0.5–0.8μm. The competitive interaction of progestins with steroid receptors may be of putative importance in explaining the progestin action at the target site.