In vivo analysis of progesterone receptor action in the uterus during embryo implantation

In order for a successful pregnancy to occur, the embryo must attach to the luminal epithelial cells and invade into the stroma. Then, the surrounding stromal cells need to undergo decidualization in order to establish the vasculature necessary for survival of the embryo. These events in early pregnancy are tightly regulated by the steroid hormones, estrogen (E2) and progesterone (P4), through their cognate receptors, the estrogen receptor (ER) and the progesterone receptor (PR), respectively. Using a mouse model in which the PR has been ablated, it was demonstrated that the PR is necessary for embryo implantation and decidualization. Therefore, understanding the mechanism of PR action in the adult uterus is necessary in order to understand the events of early pregnancy. Insights from both mouse models and human samples have been integral in elucidating uterine PR action. These studies have shown that not only PR target genes, but also mediators of PR action are important for correct PR action in early pregnancy. Many of the genes involved in PR action in early pregnancy have also been shown to have roles in uterine diseases such as endometriosis and endometrial cancer. Therefore, the integration of mouse and human studies on PR action in the uterus will be important for the future understanding of uterine diseases and in the development of treatment for these diseases.     

In vivo strain gage implantation in rats

A technique for the fabrication of encapsulated micro-miniature rosette strain gages for in vivo implantation is described. The gage units have an overall area of ten square millimeters (2.5 mm × 4.0 mm), and hence can be installed in very small experimental animals, particularly rodents. Using a rat model, strain data for up to 12 days have been obtained and in vitro studies have validified the in vivo strain recordings.     

Molecular mechanisms involved in progesterone receptor regulation of uterine function

The ovarian steroid hormone progesterone is a major regulator of uterine function. The actions of this hormone is mediated through its cognate receptor, the progesterone receptor, Pgr. Ablation of the Pgr has shown that this receptor is critical for all female reproductive functions including the ability of the uterus to support and maintain the development of the implanting mouse embryo. High density DNA microarray analysis has identified direct and indirect targets of Pgr action. One of the targets of Pgr action is a member of the Hedgehog morphogen Indian Hedgehog, Ihh. Ihh and members of the Hh signaling cascade show a coordinate expression pattern in the mouse uterus during the preimplantation period of pregnancy. The expression of Ihh and its receptor Patched-1, Ptc1, as well as, down stream targets of Ihh-Ptch1 signaling, such as the orphan nuclear receptor COUP-TF II show that this morphogen pathway mediates communication between the uterine epithelial and stromal compartments. The members of the Ihh signaling axis may function to coordinate the proliferation, vascularization and differentiation of the uterine stroma during pregnancy. This analysis demonstrates that progesterone regulates uterine function in the mouse by coordinating the signals from the uterine epithelium to stroma in the preimplantation mouse uterus.     

In vivo inhibition of epidermal growth factor receptor autophosphorylation prevents receptor internalization

The question whether epidermal growth factor (EGF)-induced receptor endocytosis requires the prior autophosphorylation via the EGF receptor (EGFR) kinase domain has been a matter of long-standing debate. In the airway epithelial cell line NCI-H292, the EGFR kinase domain inhibitor BIBW 2948 BS was found to inhibit both autophosphorylation and subsequent internalization of the endogenous EGFR with similar IC₅₀ values. Applying an ex vivo EGFR internalization assay in a clinical study, the in vivo effect of inhalatively administered BIBW 2948 BS was determined directly at the targeted receptor in airway tissues from COPD patients. In these experiments, the in vivo inhibition of the EGFR kinase domain prevented the EGF-induced internalization of EGFR.     

Expression of PIK3IP1 in the murine uterus during early pregnancy

The ovarian steroid hormones, estrogen (E2) and progesterone (P4), are essential regulators of uterine functions necessary for development, embryo implantation, and normal pregnancy. ARID1A plays an important role in steroid hormone signaling in endometrial function and pregnancy. In previous studies, using high density DNA microarray analysis, we identified phosphatidylinositol-3-kinase interacting protein 1 (Pik3ip1) as one of the genes up-regulated by ARID1A. In the present study, we performed real-time qPCR and immunohistochemistry analysis to investigate the regulation of PIK3IP1 by ARID1A and determine expression patterns of PIK3IP1 in the uterus during early pregnancy. The expression of PIK3IP1 was strong at the uterine epithelial and stromal cells of the control mice. However, expression of PIK3IP1 was remarkably reduced in the Pgr^cre/+Arid1a^f/f mice and progesterone receptor knock-out (PRKO) mice. During early pregnancy, PIK3IP1 expression was strong at day 2.5 of gestation (GD 2.5) and then slightly decreased at GD 3.5?at the epithelium and stroma. After implantation, PIK3IP1 expression was detected at the secondary decidualization zone. To determine the ovarian steroid hormone regulation of PIK3IP1, we examined the expression of PIK3IP1 in ovariectomized control, Pgr^cre/+Arid1a^f/f, and PRKO mice treated with P4 or E2. P4 treatment increased the PIK3IP1 expression at the luminal and glandular epithelium of control mice. However, the PIK3IP1 induction was decreased in both the Pgr^cre/+Arid1a^f/f and PRKO mice, compared to controls. Our results identified PIK3IP1 as a novel target of ARID1A and PGR in the murine uterus.     

Inhibition of prostaglandin F2alpha synthesis and oxytocin receptor by progesterone antagonists in bovine endometrial cells in vitro

Oxytocin receptor (OTR) expression is suppressed by progesterone (P4) during the luteal phase of the estrous cycle and then it increases at the time of luteolysis, but its regulation is still not completely understood. In vitro studies to determine the mechanism of action are hindered because OTR spontaneously upregulates in vitro and it is impossible to alter expression with P4 or estradiol. During recent studies examining the effect of P4 and an antagonist (mifepristone) on PG secretion, we found that mifepristone attenuated OT-stimulated PG secretion from endometrial epithelial cells. The objective of the present study was to determine, whether this effect of mifepristone was due to changes in prostaglandin synthesis and/or OTR. A time-course showed that mifepristone (5 microM) had no significant effect after 24 h but by 72 h it decreased PGF(2alpha) secretion (P<0.01) and abolished the response of the cells to OT (P<0.01). The presence or absence of P4 did not affect the response to mifepristone. To determine the site of action of mifepristone, cells were cultured for 72 h with or without mifepristone and then COX-1 and COX-2 were measured by Western blotting and OTR was measured by saturation analysis. The results showed that mifepristone did not affect basal or PMA-stimulated expression of either COX-1 or COX-2 but did, however, decrease OTR number (P<0.05). These data demonstrate that OTR and the response to OT can be downregulated in endometrial epithelial cells in vitro via a mechanism involving the P4 receptor.     

pNEgr-mIL-12真核表达载体的体外和体内生物学活性检测

肿瘤的基因-放射治疗是近年来发展起来的新技术.分别于体外和体内检测含辐射敏感启动子和mIL-12基因的真核表达载体（pNEgr-mIL-12）的生物学活性.体外经酶联免疫吸附试验(enzyme linked immunosorbent assay, ELISA)法检测转染pNEgr-mIL-12重组质粒的COS-7和B16细胞可经辐射诱导mIL-12 p70表达,于1.5～2.0 Gy照射后表达增高最明显,COS-7细胞于照后4 h 达峰值,B16细胞的表达水平随照射后时间延长而逐渐增高;pNEgr-mIL-12重组质粒联合电离辐射治疗小鼠移植肿瘤,单次或多次注射pNEgr-mIL-12重组质粒,联合局部照射能够抑制小鼠移植肿瘤生长,与单纯照射组比较肿瘤生长速度减慢,瘤重降低,尤以多次给予质粒治疗组效果明显.为进一步探讨最佳治疗方案及临床肿瘤病人的基因放射治疗提供了初步依据.     

Progesterone receptors in the human uterus and their possible role in parturition

An overview is given on the role of progesterone in parturition in the human. Progesterone withdrawal is considered to be a major event for the beginning of parturition. However, in the human, no evidence exists in favour of a decline in placental progesterone production prior to labour. Progesterone actions are mediated by two functionally different but structurally highly related intranuclear proteins, progesterone receptor (PR) A and PRB. In the human, functional progesterone withdrawal is thought to play a role. This may be mediated by a change in the expression of the two isoforms of the PR, with an increase in the PRA:PRB ratio, and this is accompanied by an increase in the expression of the estrogen receptor. These mechanisms are considered to be critical for the endocrine control of parturition.     

Differential expression and anti-oxidant function of glutathione peroxidase 3 in mouse uterus during decidualization

Glutathione peroxidase 3 (GPX3) is an important member of antioxidant enzymes for reducing reactive oxygen species and maintaining the oxygen balance. Gpx3 mRNA is strongly expressed in decidual cells from days 5 to 8 of pregnancy. After pregnant mice are treated with GPX inhibitor for 3 days, pregnancy rate is significantly reduced. Progesterone stimulates Gpx3 expression through PR/HIF1α in mouse endometrial stromal cells. In the decidua, the high level of GPX3 expression is closely associated with the reduction of hydrogen peroxide (H₂O₂). Based on our data, GPX3 may play a major role in reducing H₂O₂ during decidualization.     

Developmental profiles of glial enzymes in the chick embryo: In vivo and in culture

The activities of two glial cell enzymes, glutamine synthetase (a marker for astrocytes) and 2′,3′-cyclic nucleotide 3′-phosphohydrolase (a marker for oligodendrocytes and myelination) were studied in the developing chick embryo brain in vivo and in cultures derived from chick embryos. The in vivo findings showed that the activities of both enzymes parallel the patterns of gliogenesis and myelination. Glutamine synthetase follows similar patterns in culture and in vivo, whereas the developmental profile of 2′,3′-cyclic nucleotide 3′-phosphohydrolase appears to be affected by the culture conditions.     

Regulation of cytosol and nuclear progesterone receptors in rabbit uterus by estrogen,antiestrogen and progesterone administration

A synthetic progestin, 16α-ethyl-21-hydroxy-19-nor-4-pregnene-3,20-dione (ORG 2058), was utilized to measure progesterone receptors from the rabbit uterus. This steroid has a high affinity for both cytosol and nuclear receptors, with K_D values of 1.2 nM (at 0–4°C) and 2.3 nM (at 15°C), respectively. Administration of estradiol-17β or a non-steroidal antiestrogen, tamoxifen, for 5 days to estrous rabbits led to a progressive rise in the cytosol receptor levels: from 34 000 to 120 000 (estradiol-17β) and 80 000 (tamoxifen) receptors/ cell, without any major influence on the nuclear receptor content. A single intravenous injection of progesterone (5 mg/kg) elicited a 3-fold increase in the mean nuclear receptor content at 30 min after injection (from 18 000 to 48 000 receptors/nucleus). Nuclear receptor accumulation was short-lived and returned to control levels within 4 h after treatment. A second dose of progesterone given 24 h later doubled the nuclear receptor level (from 18 000 to 35 000 receptor/nucleus). The concomitant decline in the cytosol receptor content was twice that accounted for by the nuclear receptor accumulation (70 000 vs. 30 000, and 40 000 vs. 17 000 receptors/cell, after the first and second progesterone injection, respectively). Following progesterone administration, the cytosol receptor level reached a nadir by 30 min, exhibited minimal replenishment within the ensuing 24 h, and remained at approx. 50% of the pretreatment values. After a single dose or two consecutive doses of progesterone, total uterine progesterone receptor content declined to about 60% of the level prior to each dose, a nadir being reached at 2 h after treatment.     

A genomic approach to identify novel progesterone receptor regulated pathways in the uterus during implantation

The cellular actions of steroid hormone progesterone (P) are mediated via its nuclear receptors, which regulate the expression of specific target genes. The identity of gene networks that are regulated by the P receptors (PRs) in the uterus at various stages of the reproductive cycle and pregnancy, however, remain largely unknown. In this study, we have used oligonucleotide microarrays to identify mRNAs whose expression in the pregnant mouse uterus is modulated by RU486, a well-characterized PR antagonist, which is also an effective inhibitor of implantation. We found that, in response to RU486, expression of mRNAs corresponding to 78 known genes was down-regulated at least 2-fold in the preimplantation mouse uterus. The PR regulation of several of these genes was ascertained by administering P to ovariectomized wild-type and PR knockout (PRKO) mice. Detailed spatio-temporal analysis of these genes in the pregnant uterus indicated that their expression in the epithelium and stroma could be correlated with the expression of PR in those cell types. Furthermore, time-course studies suggested that many of these genes are likely primary targets of PR regulation. We also identified 70 known genes that were up-regulated at least 2-fold in the pregnant uterus in response to RU486. Interestingly, initial examination of a number of RU486-inducible genes reveals that their uterine expression is also regulated by estrogen. The identification of several novel PR-regulated gene pathways in the reproductive tract is an important step toward understanding how P regulates the physiological events leading to implantation.     

在体膜片钳技术的新进展

在体膜片钳是指在整体动物上直接对其中枢神经元进行全细胞膜片钳记录的技术,在生理学和药理学研究中具有良好的应用前景.常规采用的是盲法记录,最近出现的可视法记录,采用双光子靶向膜片钳（two-photon targeted patching,TPTP）技术,通过基因操作在动物脑内目标神经元中构建特异表达的荧光标志,可以做到对特定神经元亚群的靶向研究.对这两种方法的原理和操作进行了简单的介绍.     

Development of p-carborane-based nonsteroidal progesterone receptor antagonists

Progesterone receptor (PR) regulates various physiological processes, including the female reproductive system, and development of nonsteroidal PR antagonists is considered desirable for clinical application, as they are expected to have reduced side effects. We have synthesized a series of nonsteroidal PR antagonists using a 4-cyanophenyl-p-carborane core structure. Among them, compound 14d exhibited potent PR-antagonistic activity (IC₅₀: 27 nM). It showed high binding affinity for PR, but did not bind to androgen receptor or estrogen receptor. This PR-selective antagonist may be a promising lead compound for clinically applicable progesterone receptor modulators.     

In vivo measurement of shoulder joint loads during activities of daily living

Until recently the contact loads acting in the glenohumeral joint have been calculated using musculoskeletal models or measured in vitro. Now, contact forces and moments are measured in vivo using telemeterized shoulder implants. Mean total contact forces from four patients during eight activities of daily living are reported here.Lifting a coffee pot (1.5 kg) with straight arm caused an average force of 105.0%BW (%body weight) (range: 90–124.6%BW), while setting down the coffee pot in the same position led to higher forces of 122.9%BW on the average (105.3–153.4%BW). The highest joint contact forces were measured when the straight arm was abducted or elevated by 90° or more, with a weight in the hand. Lifting up 2 kg from a board up to head height caused a contact force of 98.3%BW (93–103.6%BW); again, setting it down on the board led to higher forces of 131.5%BW (118.8–144.1%BW). In contrast to previously calculated high loads, the contact force during passive holding of a 10 kg weight laterally was only 12.3%BW (9.2–17.9%BW), but when lifting it up to belt height it increased to 91.5%BW (87–95%BW).The moments transferred inside the joint at our patients varied much more than did the forces both inter and intra-individually.Our data suggest that patients with shoulder problems or during the first post-operative weeks after shoulder fractures or joint replacements should avoid certain activities encountered during daily living e.g. lifting or holding a weight with an outstretched arm. Some energy-related optimization criteria used in the literature for analytical musculoskeletal shoulder models must now be reconsidered.