Progesterone receptor membrane component-1 (PGRMC1) interacts with plasminogen activator inhibitor RNA binding protein-1 (PAIRBP1), a membrane-associated protein involved in the antiapoptotic action of progesterone (P4). In this paper, the first studies were designed to assess the ovarian expression pattern of PGRMC1 and PAIRBP1. Western blot analysis revealed that spontaneously immortalized granulosa cells (SIGCs) as well as granulosa and luteal cells express both proteins. Luteal cells were shown to express more PGRMC1 than granulosa cells. Immunohistochemical studies confirmed this and demonstrated that PGRMC1 was present in thecal/stromal cells, ovarian surface epithelial cells, and oocytes. PAIRBP1 was also expressed in thecal/stromal cells and ovarian surface epithelial cells but not oocytes. Furthermore, PAIRBP1 and PGRMC1 were detected among the biotinylated surface proteins that were isolated by avidin affinity purification, indicating that they localized to the extracellular surface of the plasma membrane. Confocal microscopy revealed that both of these proteins colocalize to the plasma membrane as well as the cytoplasm. The second studies were designed to assess PGRMC1's role in P4's antiapoptotic actions. These studies showed that overexpression of PGRMC1 increased 3H-P4 binding and P4 responsiveness. Conversely, treatment with a PGRMC1 antibody blocked P4's antiapoptotic action. Taken together, the present findings indicate that both PAIRBP1 and PGRMC1 show a similar expression pattern within the ovary and colocalize to the extracellular surface of the plasma membrane. At the plasma membrane, these two proteins interact to form a complex that is required for P4 to transduce its antiapoptotic action.

Connective tissue growth factor (CTGF), a member of the CCN family of secreted matricellular proteins, regulates fibrosis, angiogenesis, cell proliferation, apoptosis, tumor growth, and metastasis. However, the role of CTGF and its regulation mechanism in Wilms' tumor remains largely unknown. We found that the bioactive lipid sphingosine-1-phosphate (S1P) induced CTGF expression in a concentration-and time-dependent manner in a Wilms' tumor cell line (WiT49), whereas FTY720-phosphate, an S1P analogue that binds all S1P receptors except S1P 2 , did not. Further, the specific S1P 2 antagonist JTE-013 completely inhibited S1P-induced CTGF expression, whereas the S1P 1 antagonist VPC44116 did not, indicating that this effect was mediated by S1P 2 . This was confirmed by adenoviral transduction of S1P 2 in WiT49 cells, which showed that overexpression of S1P 2 increased the expression of CTGF. Induction of CTGF by S1P was sensitive to ROCK inhibitor Y-27632 and c-Jun NH 2 -terminal kinase inhibitor SP600125, suggesting the requirement of RhoA/ROCK and c-Jun NH 2 -terminal kinase pathways for S1P-induced CTGF expression. Interestingly, the expression levels of CTGF were decreased in 8 of 10 Wilms' tumor tissues compared with matched normal tissues by quantitative real-time PCR and Western blot analysis. In vitro, human recombinant CTGF significantly inhibited the proliferation of WiT49 cells. In addition, overexpression of CTGF resulted in significant inhibition of WiT49 cell growth. Taken together, these data suggest that CTGF protein induced by S1P 2 might act as a growth inhibitor in

The initial study was designed to determine whether all granulosa cells (GCs) undergo apoptosis in vitro. GCs were isolated from immature rat ovaries and separated on a 15-45% Percoll gradient. Twelve fractions were collected, and GCs were pooled according to size: small GCs (approximately 50 mu 2; fractions 2-5) and large GCs (> or = 75 mu 2; fractions 6-8). GCs were cultured in serum-free medium for 24 h. After 24 h of culture, fragmented DNA, detected by in situ end labeling of the 3'OH ends of DNA fragments, was observed within 70-80% of large GCs. Similarly, in situ DNA staining demonstrated that at least 50% of large GCs possessed apoptotic nuclei. These degenerative changes in DNA were observed within < or = 5% of small GCs. These studies demonstrate that in serum-free medium, most large GCs die via an apoptotic mechanism within 24 h. Subsequent studies focused on the mechanism by which epidermal growth factor (EGF) inhibits large GC apoptosis. EGF reduced the percentage of large GCs with apoptotic nuclei from 47 +/- 1% for controls to 18 +/- 2% (p < 0.05). EGF also increased progesterone (P4) secretion from large GCs (6.3 +/- 0.7 for controls vs. 18.7 +/- 1.0 ng/ml for EGF treatment; p < 0.05). The effect of EGF on apoptosis was mimicked by P4 and attenuated by the P4 antagonist, RU 486, and aminoglutethimide (AG), an inhibitor of P4 synthesis. The effect of AG was overridden by P4. Therefore, EGF reduces large GC apoptosis by stimulating P4 synthesis, with P4 mediating its action through its receptor.(ABSTRACT TRUNCATED AT 250 WORDS)

The amount of osteological variation among 11 Italian killifish Aphanius fasciatus populations was examined by the univariate and multivariate analysis of 40 morphometric and meristic variables of the skull and vertebral column. Populations were sampled in three geographically distinct areas (the Adriatic, Sardinia and Sicily). The statistical analysis confirmed that several populations were well differentiated. In particular, discriminant analysis revealed a strong discriminating power of the morphometric variables. Morphometrics of the vertebrae, bony elements of the pharyngeal jaws, supraoccipital and parasphenoid were the most important in discriminating populations. The dendrogram obtained by UPGMA cluster analysis shows the separation of the south‐eastern Sicilian populations, that of the Sardinian populations and that of the central‐northern Sicilian plus Adriatic populations, as well as the isolation of the Sicilian population from Pantano Viruca and of the Sardinian populations from Pauli Figu from all the others. The significance of the observed differentiation pattern is discussed.

The protein PAIRBP1, which was initially referred to as RDA288, is involved in mediating the antiapoptotic action of progesterone (P4) in spontaneously immortalized granulosa cells (SIGCs). The present studies were designed to assess the expression and function of PAIRBP1 in the different cell types within the immature rat ovary. Western blot analysis detected PAIRBP1 within whole-cell lysates of immature rat ovaries. Equine gonadotropin (eCG) induced a 3-fold increase in ovarian levels of PAIRBP1. Moreover, human chorionic gonadotropin (hCG), given 48 h after eCG, maintained these elevated levels for up to 4 days. Immunohistochemical analysis confirmed this and further demonstrated that interstitial, thecal, and surface epithelial cells also expressed PAIRBP1. The level of PAIRBP1 in these cells was not influenced by gonadotropin treatment. In contrast, eCG stimulated an increase in PAIRBP1 within the granulosa cells of the developing follicles. Treatment with hCG induced ovulation and ultimately the formation of corpora lutea (CL). High levels of PAIRBP1 expression were also observed within the luteal cells. Immunocytochemical studies on living, nonpermeabilized granulosa and luteal cells revealed that some PAIRBP1 localized to the extracellular surface of these cells. The presence of PAIRBP1 on the extracellular surface was consistent with the observation that an antibody to PAIRBP1 attenuated P4's antiapoptotic action in both granulosa and luteal cells. Although the PAIRBP1 antibody attenuated P4's action, it did not reduce the capacity of cells to specifically bind (3)H-P4. Immunoprecipitation with the PAIRBP1 antibody pulled down the membrane P4 binding protein known as progesterone receptor membrane complex-1 (PGRMC1; rat homolog accession number AJ005837). Taken together, these findings suggest that gonadotropins regulate the expression of PAIRBP1 in granulosa and luteal cells and that PAIRBP1 plays an important role in mediating P4's antiapoptotic action in these ovarian cell types. The exact mechanism of PAIRBP1's action remains to be elucidated, but it may involve an interaction with PGRMC1.

New asymmetric (salen)Mn III and UO 2 complexes containing a calix [4]arene unit in the ligand framework were synthesized. The UO 2 complexes were characterized by 1 H-, 13 C-, 2D TOCSY and T-ROESY NMR spectroscopy. Furthermore, the structure of one UO 2 complex was determined by singlecrystal X-ray analysis. The data showed that UO 2 complexes, which can be considered in first approximation models of the Mn=O oxidant active species, possess a chiral pocket and

Progesterone (P4) inhibits small granulosa cell (GC) mitosis and large GC apoptosis. These actions are steroid specific and dose dependent and are inhibited by the progesterone receptor (PR) antagonist, RU-486. However, these cells do not express the nuclear PR but rather an ill-defined P4-binding protein (P4BP). This binding protein could function as a receptor and mediate P4's actions in GCs. Therefore, a series of studies was designed to characterize this P4BP. First, an antibody directed against the ligand-binding site of the nuclear PR was used in a Western blot analysis. This analysis revealed the presence of a 60-kDa P4BP within ovarian and GC lysates as well as within an ovarian membrane preparation. This protein was not observed in lysates of cells derived from the ovarian surface epithelium. In addition, this P4BP was immunoprecipitated by an antibody to the alpha1 chain of the gamma aminobutyric acidA (GABA(A)) receptor, suggesting that the P4BP could be the ovarian GABA(A) receptor. Since activation of the rat ovarian GABA(A) receptor increases intracellular cAMP levels, GCs were cultured with control medium supplemented with either 8-bromo-cAMP (8-br-cAMP), P4, or muscimol (a GABA agonist). Increases in cAMP were detected by monitoring the cAMP-dependent phosphorylation of cAMP response element-binding protein (CREB). Phosphorylated CREB was not observed in control or P4-treated cultures, but it was detected in the majority of both small and large GCs exposed to either 8-br-cAMP or muscimol. Since activation of the GABA(A) receptor with muscimol increases phosphorylated CREB but P4 does not, this study indicates that P4 does not activate the ovarian GABA(A) receptor. However, both bicuculline, a GABA(A) receptor antagonist, and the antibody to PR inhibited P4's ability to prevent both insulin-dependent mitosis and apoptosis. Collectively, these studies suggest that P4 mediates its anti-mitotic and anti-apoptotic effects through this 60-kDa P4BP, which has GABA(A) receptor-like properties and is localized within the surface membrane of GCs.

Progesterone (P(4)) inhibits granulosa cell apoptosis in a steroid-specific, dose-dependent manner, but these cells do not express the classic nuclear P(4) receptor. It has been proposed that P(4) mediates its action through a 60-kDa protein that functions as a membrane receptor. The present studies were designed to determine the P(4) binding characteristics of this protein. Western blot analysis using an antibody that recognizes the P(4) binding site of the nuclear P(4) receptor (C-262) confirmed that the 60-kDa protein was localized to the plasma membrane of both granulosa cells and spontaneously immortalized granulosa cells (SIGCs). To determine whether this protein binds P(4), proteins were immunoprecipitated with the C-262 antibody, electrophoresed, transferred to nitrocellulose, and probed with a horseradish peroxidase-labeled P(4) in the presence or absence of nonlabeled P(4). This study demonstrated that the 60-kDa protein specifically binds P(4). Scatchard plot analysis revealed that (3)H-P(4) binds to a single site (i.e., single protein), which is relatively abundant (200 pmol/mg) with a K(d) of 360 nM. (3)H-P(4) binding was not reduced by dexamethasone, mifepristone (RU 486), or onapristone (ZK98299). Further studies with SIGCs showed that P(4) inhibited apoptosis and mitogen-activated protein kinase kinase (MEK) activity, and maintained calcium homeostasis. These studies taken together support the concept that the 60-kDa P(4) binding protein functions as a low-affinity, high-capacity membrane receptor for P(4).