Modulation of the progesterone receptor in the fetal uterus of the progesterone-primed guinea pig in vivo and in organ culture

Guinea pig fetuses were treated with progesterone for 7 days before placing fetal uteri in organ culture to see if progesterone pre-treatment of fetuses in utero would permanently inhibit the spontaneous rise in progesterone receptor which occurs in organ culture. The data show that: the basal level of progesterone receptor in fetal uteri was not affected by the progesterone treatment and progesterone receptor concentrations in vitro were also not inhibited. When guinea pig fetuses were treated sequentially with progesterone and estradiol, estradiol failed to provoke an uterotrophic effect but it retained its ability to stimulate progesterone receptor concentrations.     

Studies with antibodies against the conserved cysteine region of progesterone receptor

Polyclonal antibodies were generated against two synthetic peptides corresponding to sequences from the DNA-binding domain of steroid receptors. The sequence for peptide 1 (13 amino acids) lies between the two putative metal-binding loops of the conserved cysteine region while the sequence for peptide 2 (12 amino acids) lies within one loop. Peptide antibodies were generated by injecting rabbits with peptide conjugated to bovine serum albumin. By Western blot analysis, antibodies to peptide 2 recognized chick and human progesterone receptor and human glucocorticoid receptor, but peptide 1 antibodies did not. No cross-reactivity with native chick progesterone receptor was detected with either anti-peptide. These findings suggest that the epitopes for peptide 2 antibodies, and possibly for peptide 1 antibodies, are inaccessible to antibody in the native receptor.     

The rabbit progesterone receptor. Evidence for a single steroid-binding subunit and characterization of receptor mRNA

Monoclonal antibodies were used to study the structure and the biosynthesis of the rabbit progesterone receptor. Proteins in nonfractionated uterine cytosol were submitted to gel electrophoresis in denaturing conditions, transferred onto nitrocellulose, and reacted with monoclonal antireceptor antibodies and 125I-protein A. A single 110,000-dalton protein was observed when precautions were taken during homogenization of the uteri and protease inhibitors used. Smaller forms of receptor (essentially of 79,000 daltons but also of 72,000 and in some experiments of 64,000 daltons) were present when these precautions were not observed and thus probably arose from artifactual proteolysis of receptor. When poly(A)+ RNA from rabbit uterus was translated in a reticulocyte lysate and the radioactive proteins precipitated by the antireceptor monoclonal antibodies, a radioactive protein of 110,000 daltons was also observed. Further evidence that this protein was the product of the translation of progesterone receptor mRNA was obtained by precipitation and immunoaffinity purification with several antireceptor monoclonal and polyclonal antibodies, inhibition of immunoprecipitation by purified receptor and its absence in a receptor-poor tissue (liver). Estrogen treatment is known to increase the concentration of progesterone receptor. RNA translation experiments showed that this effect is due to an increase in the concentration of receptor mRNA. The size of this messenger RNA was studied by sucrose gradient ultracentrifugation, followed by mRNA translation, and specific immunoprecipitation: progesterone receptor mRNA was found by this method to sediment at 20 S.     

Characteristics of the nuclear translocation of progesterone receptor in fetal guinea pig uterus "in vivo", "in vitro" and in organ culture

The translocation of progesterone receptor from the cytosol into the nucleus was studied under "in vivo" and "in vitro" conditions in the uteri of guinea pig fetuses exposed to progesterone or a synthetic progestin, R5020. Progesterone treatment of estrogen-primed fetuses leads to a rapid (before 1h) transfer of cytosol progesterone receptor into the nucleus which is, however, short-lived (less than 3h). A rapid decrease in the retention of the estrogen receptor in the nucleus also occurs. In the "in vitro" incubations of whole fetal uteri, translocation of progesterone receptor is temperature-dependent and specific for progesterone and R5020; estradiol and cortisol have no effect. Putative progesterone receptors can also be induced in explants of fetal guinea pig uteri in organ culture which translocate from the cytosol into the nucleus under the same "in vitro" conditions as in whole uteri. Fetal uterine progesterone receptor, either stimulated "in vivo" by estrogen-priming or induced in organ culture, translocates from the cytosol into the nucleus and this process seems to be accompanied by a decrease in retention of the estrogen receptor in the nucleus which appears to be the mechanism by which progesterone antagonises estrogen action in fetal guinea pig uterus.     

孕酮受体在雌性小鼠生殖系统中的分布及RU486对其影响

用免疫细胞化学ＬＳＡＢ法,兔抗人孕酮受体抗体（适用于小鼠）显示小鼠卵巢、输卵管、子宫和阴道中的孕酮受体。结果显示：未注射ＲＵ４８６的动物的子宫与阴道内孕酮受体丰富,输卵管中含量较少,卵巢中未见阳性反应物质。ＲＵ４８６注射４天后,子宫与阴道中孕酮受体明显减少;注射后７天,输卵管、子宫和阴道都为阴性反应。该结果显示了孕酮受体在雌性小鼠生殖系统内的分布特点,提示ＲＵ４８６封闭孕酮受体的最佳时间为用药后第７天。     

Lysophosphatidic receptor, LPA3, is positively and negatively regulated by progesterone and estrogen in the mouse uterus

Reciprocal interactions between blastocysts and receptive uteri are essential for successful implantation. This process is regulated by the timely interplay of two ovarian hormones, progesterone and estrogen. However, the molecular targets of these hormones are largely unknown. We showed recently that a small bioactive lysophospholipid, lysophosphatidic acid, plays a pivotal role in the establishment of implantation via its cellular receptor, LPA(3). Here we demonstrate that LPA(3) expression is positively and negatively regulated by steroid hormones in mouse uteri. The LPA(3) mRNA level in the uteri increased during early pseudopregnancy, peaking around 3.5 days post coitus (3.5 d.p.c.), then, decreased to the basal level on 4.5 d.p.c. LPA(3) expression remained at a low level in ovariectomized mice, and administration of progesterone to ovariectomized mice up-regulated LPA(3) mRNA expression. In addition, simultaneous administration of estrogen counteracted the effect of progesterone. These results show that progesterone and estrogen cooperatively regulate LPA(3) expression, thereby contributing to the receptivity of uteri during early pregnancy.     

Expression of alpha and gamma interferon receptors in the sperm cell

The present study was conducted to examine whether or not the sperm cell has the expression of receptors for interferon (IFN) -alpha and -gamma. This was investigated using specific antibodies. Antibody to IFN-alpha receptor reacted with the acrosomal and tail regions of the murine sperm cell in the indirect immunofluorescence technique (IFT) and immunoscanning electron microscopic procedure (ISEP). In the immunoprecipitation and Western blot procedures, this antibody specifically recognized a protein band of approximately 100 kD, which corresponds to the molecular weight of IFN-alpha receptor present in other cell types. Antibody to IFN-gamma receptor specifically reacted with the posterior head, midpiece, and tail regions of sperm cell in IFT and ISEP, and recognized a band of approximately 85 kD in the immunoprecipitation and Western blot procedures, corresponding to the IFN-alpha receptor. Similar bands of approximately 100 kD and approximately 85 kD molecular identities were also detected in the testes extracts and sperm extracts of other mammalian species namely human, rabbit, and pig, the species tested. These findings indicate that the mammalian sperm cell has expression of IFN-alpha and IFN-gamma receptors, which seem to develop during spermatogenesis in the testes. These findings may have implications in male infertility and antisperm contraceptive vaccine development.     

Spatial and molecular aspects of estrogen and progesterone receptor expression in human uteri and uterine carcinomas

The expression of steroid hormone receptors as molecules reflecting processes of development and differentiation in the human uterine tissue was analysed in a spatial distinct fashion: tissue samples were excised at the fundus and at different, spatially distinct positions of the uterus. They were analysed for concentrations of cytosolic estrogen and progesterone receptors in supernatants from frozen sections using an isoelectric focusing technique. The spatial and molecular distinct, qualitative and quantitative pattern of their expression in the human uterus and uterine adenocarcinomas were studied by sectioning tissue sample from the functionalis through the basalis of the endometrium until reaching deep myometrial parts of the tissue: (1) Specific spatial patterns of estrogen and progesterone receptor levels were detectable throughout the menstrual cycle. (2) For proliferative endometrium from the functionalis to the basalis of the endometrium, the content of both cytosolic receptor species increased up to 6-fold. (3) Differences detectable were less pronounced in the myometrial part of the tissue. (4) Differences of steroid receptor concentrations measured in the endometrium at different uterine positions were highest between fundus and corpus of the endometrium. (5) Maximal differences were detectable around ovulation. (6) After secretory transformation of the organ, specific patterns were still detectable, however quantitative differences were less pronounced. (7) Additionally, quantitative differences measurable were accompanied by variations of molecular properties of the progesterone receptor as demonstrated in an isoelectric focusing gel. (8) In endometrial adenocarcinomas, not only significant quantitative alterations in steroid receptor content were measured, but also a significantly changed spatial pattern of receptor concentrations, also a change of the molecular properties of the progesterone receptor was resolved if these tumor parameters were compared to those detected in the normal tissue of the same organ surrounding the tumor.     

Estrogen-alpha and progesterone receptor expression in cystic endometrial hyperplasia and pyometra in the bitch

Estrogen-alpha receptor (ER) and progesterone receptor (PR) were examined immunohistochemically in uteri of normal bitches, in uteri of bitches with cystic endometrial hyperplasia-mucometra (CEH-M) and in uteri of bitches with endometritis-pyometra (E-P), under exogenous progesterone treatment.In the CEH-M group, the ER- and PR-scores of all uterine cell types were higher than the ER- and PR-scores of normal uteri, although these differences were not always statistically significant. The ER-scores of E-P group were significantly lower than the ER-scores of the normal uteri and CEH-M group. The PR-scores of the E-P group tended to be higher than the PR-scores of the normal uteri, except for the surface epithelium, although these differences were not statistically significant. Exogenous progesterone treated bitches with CEH-M or E-P showed reduced ER- and PR-scores in the different uterine cell types, compared with the corresponding nontreated CEH-M or E-P group.The differences in ER and PR expression between CEH-M and E-P suggest different factors in the pathogenesis of both entities. Although, these changes in ER and PR expression do not seem to be directly involved in the pathogenesis of CEH-M and E-P. It is suggested that for CEH-M and progestin induced CEH-M a hormone dependent pathway is responsible. For P, the trigger may be bacterial infection.     

Progesterone receptors on human spermatozoa

Progesterone, primarily recognized as a female steroid hormone, is reported to affect several sperm functions especially capacitation, motility and acrosome reaction. These effects of progesterone on the spermatozoa are mediated via the progesterone binding sites/progesterone receptor (PR) on the acrosomal membrane. These receptors in response to progesterone increase the intercellular Ca2+ levels and stimulate Ca2+ influx in the mature human spermatozoa via non-genomic mode of actions. Characterization of this receptor reveals that the sperm PR is masked protein and is exposed to the surface by some non-ionic detergents. Localized on to the acrosome region of the spermatozoa, these receptors are recognized by most antibodies directed towards the C-terminal region of the conventional PR. The estimated molecular weight of PR on spermatozoa varies from 27 kDa to 85 kDa. At the molecular level, sequences encoding for the entire DNA and hormone binding domains of the conventional PR are detected in the mRNA derived from spermatozoa. No insertions, deletions or mutations are detected in this region. These results are suggestive of the fact that at least the C terminal region of the conventional PR is conserved in the sperm. It is hypothesized that post-translational modifications or peptide splicing of the conventional PR in spermatozoa may possibly lead to the variant of the steroid hormone receptor. Detailed characterization of the sperm PR will be important in understanding the alternate non-genomic mode of action of steroid hormone receptors.     

有关检测雌激素、孕激素、雌激素受体和孕激素受体水平的技术性探讨

应用兔抗雌二醇抗体、兔抗孕酮抗体,鼠抗雌激素受体和鼠抗孕激素受体抗体来检测乳腺癌等病例,结果显示,兔抗雌二醇抗体和兔抗孕酮抗体所标记的病例的阳性物主要见于癌细胞胞浆,应用石蜡切片且不经任何方法处理的结果为最好。应用鼠抗雌激素受体抗体和鼠抗孕激素受体抗体和鼠抗孕激素受体抗体的病例,阳性物见于癌细胞的胞核上,但需要用隔水热抗原修复法经较长时间的处理后,方能获得最佳结果。     

Complete amino acid sequence of the human progesterone receptor deduced from cloned cDNA

A lambda gt10 library containing DNAs complementary to messenger RNAs from human breast cancer T47-D cells was constructed and screened with a cDNA probe encoding the rabbit progesterone receptor. Four overlapping clones have been sequenced. The open reading frame corresponds to a protein of 933 amino acids with a molecular weight of 98,868 Da. The cysteine rich basic region supposed to be involved in DNA binding is completely homologous in the human and rabbit receptors, whereas the C-terminal end, where hormone binding is thought to take place, differs by a single amino acid change. The human progesterone receptor is characterized, as is the rabbit receptor, by the very high proline content of its N-terminal region. When mRNAs from either human breast cancer cell lines T47-D and MCF-7 or from normal human uterus tissue were blotted and probed with the cloned cDNA, four main bands were observed (5100, 4300, 3700, and 2900 nucleotides).     

Immunohistochemical analysis of estrogen receptors in cystic-endometritis-pyometra complex in the bitch

Monoclonal antibodies to human estrogen receptor were used with an indirect immunohistochemical technique to localize in formalin-fixed, paraffin-embedded sections of the genital tract of the bitch. The presence of estrogen receptors in the uterus of dogs with cystic hyperplasia endometritis complex (CHE) was compared with normal uteri from dogs at the same stage of estrous cycle. Lower estrogen receptor expression was found in the squamous metaplastic epithelium covering the luminal surface of the endometrium from dogs with CHE than in the columnar epithelium from normal dogs. In contrast, the basal glands from uteri of CHE group dogs contained more estrogen receptor than glands from normal dogs at the same stage of estrous cycle. This was most pronounced and statistically significant in the late secretary stage of the estrous cycle. When the glands presented cystic degeneration, the estrogen receptor labeling was less pronounced. The highest estrogen receptor score was present in normal glands from dogs treated with progestins. In this group even cystic degenerated glands contained high estrogen receptor concentrations compared with those of the other groups. From this study it was concluded that the down regulation of estrogen receptor expression in the endometrial glands under the influence of rising progesterone concentrations is defective in dogs with CHE. Therefore it is suggested that the regulation of estrogen receptor expression in endometrial glands may play an important role in the pathogenesis of CHE in the bitch.     

Characterization of rat Leydig cell gonadotropin receptor structure by affinity cross-linking

The present study was intended to examine the structure of the rat Leydig cell gonadotropin receptor. Leydig cell suspensions were prepared by either collagenase digestion or mechanical disruption of the testes. The cells were incubated with 125I-human chorionic gonadotropin (hCG) following which the bound 125I-hCG was covalently cross-linked to the cell surface receptor using a cleavable (dithiobis(succinimidyl propionate] and a noncleavable (disuccinimidyl suberate) cross-linking reagent. The extracted cross-linked membrane proteins were resolved on sodium dodecyl sulfate-polyacrylamide gel electrophoresis under reducing and nonreducing conditions and subjected to autoradiographic analysis. Under nonreducing conditions, three radiolabeled bands, in addition to intact hCG and its alpha-subunit, were detected with apparent molecular weights of 184,000, 136,000, and 103,000. However, under reducing conditions, three radiolabeled bands migrated on the gel corresponding to molecular weights of 144,000, 106,000, and 75,000. The binding of 125I-hCG to the receptor was inhibited by hCG and luteinizing hormone, but not by a number of other peptides or proteins. The radiolabeled bands were not detectable in hCG down-regulated Leydig cells. Furthermore, a similar autoradiographic pattern of 125I-hCG-linked complexes was seen when the 125I-linked receptor complex was subjected to immunoprecipitation with anti-hCG antibodies followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. In addition, evidence was obtained indicating that these three labeled bands were derived from the same molecular species. The data suggests that the hCG receptor in Leydig cell is probably an oligomeric complex with a molecular weight of about 250,000, which is composed of three polypeptide chains of molecular weights 121,000, 83,000, and 52,000 held together through noncovalent forces. Additionally, collagenase treatment of Leydig cells does not appear to alter the autoradiographic pattern of the 125I-hCG-linked receptor.     

Species and tissue distribution specificity of proteins binding 16alpha,17alpha-cycloalkane derivatives of progesterone

The binding of [3H]progesterone and [3H] 16 alpha,17 alpha-cycloalkanoprogesterones to proteins from rat, rabbit, and human uteri and other organs was studied. We found that 16 alpha,17 alpha-cycloalkanoprogesterone derivatives display affinities for the uterine progesterone receptors comparable with that of the natural hormone and no substantial species differences in the affinity. Rabbit uterus was found to have no proteins distinct from the progesterone receptor that specifically bind [3H] 16 alpha,17 alpha-cycloalkanoprogesterones. At the same time, in the human uterus, we found another protein that binds some of these progesterone derivatives; it turned out to be similar to the protein from rat uterus. A similar protein with the same selectivity and affinity for steroids was also found in rat and human kidneys. Blood serum, liver, lung, and a number of other tissues were found to contain a protein of the third type that binds the same 16 alpha,17 alpha-cycloalkanoprogesterones and exhibits submicromolar Kd values for these steroids and a very low affinity for progesterone. We speculated that the introduction of a bulky substituent adjacently to the 17 beta-side chain of progesterone could result in a change in the general biodynamics of the derivative including its transport, uptake, and accumulation in tissues, which may determine the selectivity of its effect.     

Mechanisms regulating norgestomet inhibition of endometrial gland morphogenesis in the neonatal ovine uterus

In many species, endometrial gland adenogenesis occurs neonatally in an ovary- and steroid-independent manner. Chronic exposure of the developing neonatal ovine uterus to norgestomet (NOR) from birth permanently ablates endometrial gland morphogenesis or adenogenesis, creating an adult ovine uterine gland knockout (UGKO) phenotype. This study was conducted to determine the mechanism(s) whereby NOR inhibits adenogenesis in the neonatal ewe. Ewe lambs received no implant or a NOR implant at birth and on postnatal day (PND) 14, and they were necropsied on PND28. Histological analyses of the tracts indicated NOR exposure specifically inhibited endometrial adenogenesis, but no histoarchitectural differences were observed in the oviduct, cervix, or vagina. No effect of NOR treatment was detected on proliferating cell nuclear antigen (PCNA) expression in the endometrial luminal epithelium (LE), stroma, or myometrium. In control (CX) ewes, estrogen receptor alpha (ER-alpha) and progesterone receptor (PR) mRNA and protein were expressed strongly in nascent and proliferating glandular epithelium (GE) but were undetected in epithelium of NOR uteri. Expression of c-met and fibroblast growth factor receptor 2IIIb (FGFR2IIIb) mRNA was detected in the LE and GE of CX uteri. In NOR uteri, c-met was expressed in the LE similar to CX uteri, but FGFR2IIIb mRNA levels were lower than in the LE of CX uteri. Uterine hepatocyte growth factor (HGF), the ligand for c-met, and FGFR2IIIb mRNA expression was substantially lower in NOR ewes, but expression of FGF-7 and FGF-10 mRNAs, ligands for FGFR2IIIb, was unaffected. These results indicate that NOR disrupts endometrial adenogenesis by ablating epithelial ER-alpha expression and altering expression of paracrine growth factors and/or receptors involved in epitheliomesenchymal interactions. Likewise, these mechanisms are proposed to be important regulators of normal uterine gland morphogenesis in the neonate.     

High affinity progesterone binding sites of human uterine microsomal membranes

Microsomal membranes sedimented at 40 000 g were prepared from human myometrium samples. The progesterone binding properties of microsomal suspensions were determined by incubating microsomes and [3H]progesterone at 4 degrees C. Dextran-coated charcoal was used for the separation of bound and free steroids. Membrane-associated progesterone binding sites of high affinity were identified in microsomes prepared from pregnant and nonpregnant uteri. The binding was saturable (Kd approximately 4 X 10(-9) M, concentration of binding sites 400-900 fmol/mg microsomal protein) and specific for natural progesterone. Of 21 steroids tested only 21-hydroxy-4-pregnene-3,20-dione, 17 alpha-hydroxyprogesterone and testosterone showed moderate competition against progesterone with relative affinities between 7.0-20.0% (R.A. of progesterone 100%). 5 alpha-Dihydroprogesterone and 5 alpha-dihydrotestosterone showed weak cross reaction (relative affinities 2.5 and 2.0%, respectively). Corticosteroids, estrogens and the 5 synthetic progestins tested showed only weak competition with relative affinities lower than 1.0%. These microsomal progesterone binding sites of high affinity and limited capacity resemble steroid hormone receptors but they are different from the soluble cytosolic progesterone receptor of human uterus in terms of steroid specificity. The physiological function of this microsomal progesterone receptor is unknown.