Hyperglycaemic conditions perturb mouse oocyte in vitro developmental competence via beta-O-linked glycosylation of heat shock protein 90.

STUDY QUESTION: What is the effect of beta-O-linked glycosylation (O-GlcNAcylation) on specific proteins in the cumulus-oocyte complex (COC) under hyperglycaemic conditions? SUMMARY ANSWER: Heat shock protein 90 (HSP90) was identified and confirmed as being O-GlcNAcylated in mouse COCs under hyperglycaemic conditions (modelled using glucosamine), causing detrimental outcomes for embryo development. WHAT IS KNOWN ALREADY: O-GlcNAcylation of proteins occurs as a result of increased activity of the hexosamine biosynthesis pathway, which provides substrates for cumulus matrix production during COC maturation, and also for O-GlcNAcylation. COCs matured under hyperglycaemic conditions have decreased developmental competence, mediated at least in part through the mechanism of increased O-GlcNAcylation. STUDY DESIGN, SIZE, DURATION: This study was designed to examine the effect of hyperglycaemic conditions (using the hyperglycaemic mimetic, glucosamine) on O-GlcNAc levels in the mouse COC, and furthermore to identify potential candidate proteins which are targets of this modification, and their roles in oocyte maturation. PARTICIPANTS/MATERIALS, SETTING, METHODS: COCs from 21-day-old superovulated CBA × C57BL6 F1 hybrid female mice were matured in vitro (IVM). Levels of O-GlcNAcylated proteins, HSP90 and O-GlcNAc transferase (OGT, the enzyme responsible for O-GlcNAcylation) in COCs were measured using western blot, and localization observed using immunocytochemistry. For glycosylated HSP90 levels, and to test OGT-HSP90 interaction, immunoprecipitation was performed prior to western blotting. Embryo development was assessed using in vitro fertilization and embryo culture post-maturation. MAIN RESULTS AND THE ROLE OF CHANCE: Addition of the hyperglycaemic mimetic glucosamine to IVM medium for mouse COCs increased detectable O-GlcNAcylated protein levels (by western blot and immunocytochemistry), and this effect was reversed using an OGT inhibitor (P < 0.05). HSP90 was identified as a target of O-GlcNAcylation in the COC, and inhibition of HSP90 during IVM reversed glucosamine-induced decreases in oocyte developmental competence (P < 0.05). We also demonstrated the novel finding of an association between HSP90 and OGT in COCs, suggesting a possible client-chaperone relationship. LIMITATIONS, REASONS FOR CAUTION: In vitro maturation of COCs was used so that treatment time could be limited to the 17 h of maturation prior to ovulation. Additionally, glucosamine, a hyperglycaemic mimetic, was used because it specifically activates the hexosamine pathway which provides the O-GlcNAc moieties. The results in this study should be confirmed using in vivo models of hyperglycaemia and different HSP90 inhibitors. WIDER IMPLICATIONS OF THE FINDINGS: This study leads to a new understanding of how diabetes influences oocyte competence and provides insight into possible therapeutic interventions based on inhibiting HSP90 to improve oocyte quality. STUDY FUNDING/COMPETING INTEREST(S): This work was supported by a programme grant from the National Health and Medical Research Council, Australia, ID 453556. J.G.T. is a recipient of funding from and a consultant to Cook Medical Pty Ltd. The other authors have no conflicts of interest to declare.

Effect of varying glucose and glucosamine concentration in vitro on mouse oocyte maturation and developmental competence.

The effects of hyper- and hypo-glycaemic conditions during the in vitro maturation of mouse cumulus-oocyte complexes on developmental competence were examined, with an emphasis on the role of the hexosamine biosynthesis pathway. A low (1 mM) glucose concentration achieved optimal oocyte competence (3-fold higher blastocyst development rate compared with high (30 mM) glucose, P<0.05). In addition, glucose supplementation during only the first hour after release from the follicle was necessary and sufficient to support oocyte maturation and embryo development to the blastocyst stage. Glucosamine (a known hyperglycaemic mimetic and specific activator of the hexosamine pathway) was able to substitute for glucose during this first hour, indicating that flux through the hexosamine pathway is essential for oocyte competence. In the absence of glucose throughout the maturation period, glucosamine was not able to increase developmental competence, and at higher concentrations (2.5 and 5 mM) had a detrimental effect on MII and blastocyst development rates, compared with controls (P<0.05). These experiments underscore the importance of glucose metabolic pathways during in vitro maturation and support the concept that excess flux through the hexosamine pathway has detrimental consequences.

Suppressor of cytokine signaling 4 (SOCS4): moderator of ovarian primordial follicle activation.

Mammalian ovarian primordial follicle activation and regulation is considered as one of the most important stages of folliculogenesis and as such requires exquisite control. Selection of quiescent follicles to enter the growing pool determines the rate of supply of maturing follicles over the female reproductive lifespan. To coordinate this process a range of positive and negative input signals contribute to determine follicle fate. This study demonstrates that the cytokine Leukemia Inhibitory Factor (LIF) activates the Janus Kinase 1/Signal Transducers and Activators of Transcription 3 (JAK1/STAT3) signaling pathway in pre-granulosa cells and positively regulates primordial follicle activation. Negative regulation of the JAK/STAT pathway is controlled by the suppressor of cytokine signaling 4 (SOCS4) protein, which target members of negative feedback loops, Cardiotrophin like Cytokine (CLC), Poly (rC) Binding Protein 1 (PCBP1), and Cytosolic Malate Dehydrogenase (MDH1) to suppress follicle growth and development.

The effect of pressure on the crystal structure of bianthrone.

Bianthrone [10(10-oxoanthracen-9-ylidene)anthracen-9-one] consists of two tricyclic anthraceneone units connected by a carbon-carbon double bond. Crystals of the form obtained under ambient conditions are yellow and contain folded centrosymmetric conformers in which the central ring of the anthraceneone unit is non-planar. When hydrostatic pressure is applied the crystals assume a red colouration which gradually deepens as pressures increases. The colour change is limited in extent to the surface of the crystals, the bulk remaining yellow. Comparison of high-pressure, single-crystal UV-vis spectra and powder diffraction data demonstrate that the colour change is associated with the formation of a polymorph containing a conformer in which the tricyclic fragments are planar and the molecule is twisted about the central C-C bond. Single-crystal diffraction data collected as a function of pressure up to 6.5 GPa reveal the effect of compression on the yellow form, which consists of layers of molecules which stack along the [010] direction. The structure remains in a compressed form of the ambient-pressure phase when subjected to hydrostatic pressure up to 6.5 GPa, and the most prominent effect of pressure is to push the layers closer together. PIXEL calculations show that considerable strain builds up in the crystal as pressure is increased with a number of intermolecular contacts being pushed into destabilizing regions of their potentials.

Structures and aggregation of the methylamine-borane molecules, Me(n)H(3-n)N.BH(3) (n = 1-3), studied by X-ray diffraction, gas-phase electron diffraction, and quantum chemical calculations.

The structures of the molecules methylamine-borane, MeH(2)N.BH(3), and dimethylamine-borane, Me(2)HN.BH(3), have been investigated by gas-phase electron diffraction (GED) and quantum chemical calculations. The crystal structures have also been determined for methylamine-, dimethylamine-, and trimethylamine-borane, Me(n)H(3-n)N.BH(3) (n = 1-3); these are noteworthy for what they reveal about the intermolecular interactions and, particularly, the N-H...H-B dihydrogen bonding in the cases where n = 1 or 2. Hence, structures are now known for all the members of the ammonia- and amine-borane series Me(n)H(3-n)N.BH(3) (n = 0-3) in both the gas and solid phases. The structural variations and energetics of formation of the gaseous adducts are discussed in relation to the basicity of the Me(n)H(3-n)N fragment. The relative importance of secondary interactions in the solid adducts with n = 0-3 has been assessed by the semi-classical density sums (SCDS-PIXEL) approach.

Organometallic osmium(II) arene anticancer complexes containing picolinate derivatives.

Chlorido osmium(II) arene [(eta(6)-biphenyl)Os(II)(X-pico)Cl] complexes containing X = Br (1), OH (2), and Me (3) as ortho, or X = Cl (4), CO(2)H (5), and Me (6) as para substituents on the picolinate (pico) ring have been synthesized and characterized. The X-ray crystal structures of 1 and 6 show typical "piano-stool" geometry with intermolecular pi-pi stacking of the biphenyl outer rings of 6. At 288 K the hydrolysis rates follow the order 2 >> 6 > 4 > 3 > 5 >> 1 with half-lives ranging from minutes to 4.4 h illustrating the influence of both electronic and steric effects of the substituents. The pK(a) values of the aqua adducts 3A, 4A, 5A, and 6A were all in the range of 6.3-6.6. The para-substituted pico complexes 4-6 readily formed adducts with both 9-ethyl guanine (9EtG) and 9-ethyl adenine (9EtA), but these were less favored for the ortho-substituted complexes 1 and 3 showing little reaction with 9EtG and 9EtA, respectively. Density-functional theory calculations confirmed the observed preferences for nucleobase binding for complex 1. In cytotoxicity assays with A2780, cisplatin-resistant A2780cis human ovarian, A549 human lung, and HCT116 colon cancer cells, only complexes 4 (p-Cl) and 6 (p-Me) exhibited significant activity (IC(50) values < 25 microM). Both of these complexes were as active as cisplatin in A2780 (ovarian) and HCT116 (colon) cell lines, and even overcome cisplatin resistance in the A2780cis (ovarian) cell line. The inactivity of 5 is attributed to the negative charge on its para carboxylate substituent. These data illustrate how the chemical reactivity and cancer cell cytotoxicity of osmium arene complexes can be controlled and "fine-tuned" by the use of steric and electronic effects of substituents on a chelating ligand to give osmium(II) arene complexes which are as active as cisplatin but have a different mechanism of action.

Comparison of the effects of pressure on three layered hydrates: a partially successful attempt to predict a high-pressure phase transition.

We report the effect of pressure on the crystal structures of betaine monohydrate (BTM), L-cysteic acid monohydrate (CAM) and S-4-sulfo-L-phenylalanine monohydrate (SPM). All three structures are composed of layers of zwitterionic molecules separated by layers of water molecules. In BTM the water molecules make donor interactions with the same layer of betaine molecules, and the structure remains in a compressed form of its ambient-pressure phase up to 7.8 GPa. CAM contains bi-layers of L-cysteic acid molecules separated by water molecules which form donor interactions to the bi-layers above and below. This phase is stable up to 6.8 GPa. SPM also contains layers of zwitterionic molecules with the waters acting as hydrogen-bond donors to the layers above and below. SPM undergoes a single-crystal to single-crystal phase transition above 1 GPa in which half the water molecules reorient so as to form one donor interaction with another water molecule within the same layer. In addition, half of the S-4-sulfo-L-phenylalanine molecules change their conformation. The high-pressure phase is stable up to 6.9 GPa, although modest rearrangements in hydrogen bonding and molecular conformation occur at 6.4 GPa. The three hydrates had been selected on the basis of their topological similarity (CAM and SPM) or dissimilarity (BTM) with serine hydrate, which undergoes a phase transition at 5 GPa in which the water molecules change orientation. The phase transition in SPM shows some common features with that in serine hydrate. The principal directions of compression in all three structures were found to correlate with directions of hydrogen bonds and distributions of interstitial voids.

Tissue-specific progesterone receptor-chromatin binding and the regulation of progesterone-dependent gene expression.

Progesterone receptor (PGR) co-ordinately regulates ovulation, fertilisation and embryo implantation through tissue-specific actions, but the mechanisms for divergent PGR action are poorly understood. Here we characterised PGR activity in mouse granulosa cells using combined ChIP-seq for PGR and H3K27ac and gene expression microarray. Comparison of granulosa, uterus and oviduct PGR-dependent genes showed almost complete tissue specificity in PGR target gene profiles. In granulosa cells 82% of identified PGR-regulated genes bound PGR within 3 kb of the gene and PGR binding sites were highly enriched in proximal promoter regions in close proximity to H3K27ac-modified active chromatin. Motif analysis showed highly enriched PGR binding to the PGR response element (GnACAnnnTGTnC), but PGR also interacted significantly with other transcription factor binding motifs. In uterus PGR showed far more tendency to bind intergenic chromatin regions and low evidence of interaction with other transcription factors. This is the first genome-wide description of PGR action in granulosa cells and systematic comparison of diverse PGR action in different reproductive tissues. It clarifies finely-tuned contextual PGR-chromatin interactions with implications for more targeted reproductive medicine.

Outcomes of colonization with MRSA and VRE among liver transplant candidates and recipients.

Methicillin-resistant Staphylococcus aureus (MRSA) and vancomycin-resistant enterococcus (VRE) infections cause significant morbidity and mortality among liver transplant candidates and recipients. To assess rates of MRSA and VRE colonization, we obtained active surveillance cultures from 706 liver transplant candidates and recipients within 24 h of admission to an 11-bed liver transplant ICU from October 2000 to December 2005. Patients were followed prospectively to determine the cumulative risk of MRSA or VRE infection or death by colonization status. Outcomes were assessed by Kaplan-Meier survival analysis and Cox regression and multivariate logistic regression adjusting for covariates. The prevalence of newly detected MRSA nasal and VRE rectal colonization was 6.7% and 14.6%, respectively. Liver transplant candidates and recipients with MRSA colonization had an increased risk of MRSA infection (adjusted OR = 15.64, 95% CI 6.63-36.89) but not of death (adjusted OR = 1.00, 95% CI 0.43-2.30), whereas those with VRE colonization had an increased risk both of VRE infection (adjusted OR = 3.61, 95% CI 2.01-6.47) and of death (adjusted OR = 2.12, 95% CI 1.27-3.54) compared with noncolonized patients. Prevention and control strategies, including use of active surveillance cultures, should be implemented to reduce the rates of both MRSA and VRE colonization in this high-risk patient population.

The contrasting chemistry and cancer cell cytotoxicity of bipyridine and bipyridinediol ruthenium(II) arene complexes.

The synthesis and characterization of ruthenium(II) arene complexes [(eta(6)-arene)Ru(N,N)Cl](0/+), where N,N = 2,2'-bipyridine (bipy), 2,2'-bipyridine-3,3'-diol (bipy(OH)(2)) or deprotonated 2,2'-bipyridine-3,3'-diol (bipy(OH)O) as N,N-chelating ligand, arene = benzene (bz), indan (ind), biphenyl (bip), p-terphenyl (p-terp), tetrahydronaphthalene (thn), tetrahydroanthracene (tha) or dihydroanthracene (dha), are reported, including the X-ray crystal structures of [(eta(6)-tha)Ru(bipy)Cl][PF(6)] (1), [(eta(6)-tha)Ru(bipy(OH)O)Cl] (2) and [(eta(6)-ind)Ru(bipy(OH)(2))Cl][PF(6)] (8). Complexes 1 and 2 exibit CH (arene)/pi (bipy or bipy(OH)O) interactions. In the X-ray structure of protonated complex 8, the pyridine rings are twisted (by 17.31 degrees). In aqueous solution (pH = 2-10), only deprotonated (bipy(OH)O) forms are present. Hydrolysis of the complexes was relatively fast in aqueous solution (t(1/2) = 4-15 min, 310 K). When the arene is biphenyl, initial aquation of the complexes is followed by partial arene loss. Complexes with arene = tha, thn, dha, ind and p-terp, and deprotonated bipyridinediol (bipy(OH)O) as chelating ligands, exhibited significant cytotoxicity toward A2780 human ovarian and A549 human lung cancer cells. Complexes [(eta(6)-bip)Ru(bipy(OH)O)Cl] (7) and [(eta(6)-bz)Ru(bipy(OH)O)Cl] (5) exhibited moderate cytotoxicity toward A2780 cells, but were inactive toward A549 cells. These activity data can be contrasted with those of the parent bipyridine complex [(eta(6)-tha)Ru(bipy)Cl][PF(6)] (1) which is inactive toward both A2780 ovarian and A549 lung cell lines. DFT calculations suggested that hydroxylation and methylation of the bipy ligand have little effect on the charge on Ru. The active complex [(eta(6)-tha)Ru(bipy(OH)O)Cl] (2) binds strongly to 9-ethyl-guanine (9-EtG). The X-ray crystal structure of the adduct [(eta(6)-tha)Ru(bipy(OH)O)(9-EtG-N7)][PF(6)] shows intramolecular CH (arene)/pi (bipy(OH)O) interactions and DFT calculations suggested that these are more stable than arene/9-EtG pi-pi interactions. However [(eta(6)-ind)Ru(bipy(OH)(2))Cl][PF(6)] (8) and [(eta(6)-ind)Ru(bipy)Cl][PF(6)] (16) bind only weakly to DNA. DNA may therefore not be the major target for complexes studied here.

Hematopoietic Stem Cell-derived Adipocytes Promote Tumor Growth and Cancer Cell Migration.

Adipocytes, apart from their critical role as the energy storage depots, contribute to the composition of the tumor microenvironment. Our previous studies based on a single hematopoietic stem cell (HSC) transplantation model, have revealed a novel source of adipocytes from HSCs via monocyte/macrophage progenitors. Herein, we extend these studies to examine the role of HSC-derived adipocytes (HSC-Ad) in tumor progression. When cultured under adipogenic conditions, bone marrow-derived monocytic progenitors differentiated into adipocytes that accumulated oil droplets containing triglyceride. The adipokine array and ELISAs confirmed secretion of multiple adipokines by HSC-Ad. These adipocytes underwent further development in vivo when injected subcutaneously into C57Bl/6 mice. When co-injected with melanoma B16F1 cells or breast cancer E0771 cells into syngeneic C57Bl/6 mice, HSC-Ad not only accelerated both melanoma and breast tumor growth, but also enhanced vascularization in both tumors. Conditioned media from HSC-Ad supported B16F1 and E0771 cell proliferation and enhanced cell migration in vitro. Among the HSC-Ad secreted adipokines, insulin-like growth factor 1 (IGF-1) played an important role in E0771 cell proliferation. Hepatocyte growth factor (HGF) was indispensable for B16F1 cell migration, whereas HGF and platelet-derived growth factor BB (PDGF-BB) collectively contributed to E0771 cell migration. Expression levels of receptors for IGF-1, HGF, and PDGF-BB correlated with their differential roles in B16F1 and E0771 cell proliferation and migration. Our data suggest that HSC-Ad differentially regulate tumor behavior through distinct mechanisms.

The N-terminal peptide of the inhibin alpha subunit What are its endocrine and paracrine roles?

alphaN inhibin (molecular mass 23 24 kD) is present in the pro-alphaN-alphaC subunit of inhibin and can be released by cleavage at the flanking arginine residues during posttranslational processing. Although the alphaN protein isolated from bovine follicular fluid has no inhibinlike (FSH suppressing) activity, alphaN is present in high molecular weight forms of biologically active inhibin found in follicular fluid and plasma. alphaN may modify the biological activity of inhibin by influencing its half-life or access to its receptor. alphaN may also play a role in regulating fertility through a local action on ovulation by the ovary that is independent of the actions of inhibin. The evidence suggests a unique physiological significance for the precursor peptides of the inhibin-alpha subunit in both the endocrine and paracrine control of fertility.

Differentiation-dependent prolactin responsiveness and stat (signal transducers and activators of transcription) signaling in rat ovarian cells.

PRL activates an important cytokine signaling cascade that is obligatory for maintaining luteal cell function in the rat ovary. To determine when specific components of this cascade are expressed and can be activated by PRL, we analyzed the expression of receptor subtypes (short, PRL-R(s), and long, PRL-R(L)), the presence and kinetics of Stat (signal transducer and activator of transcription) activation using the PRL-response element (PRL-RE) of the alpha2M (alpha2-macroglobulin) gene, and the content and hormonal regulation of three specific modulators of cytokine signaling; the tyrosine phosphatases (SHP-1 and SHP-2), and the protein inhibitor of activated Stat3 (PIAS-3). These components were analyzed in differentiating granulosa/ luteal cells of hypophysectomized (H) rats and in corpora lutea of pregnant rats. Levels of PRL-R mRNAs increased as granulosa cells differentiated and reached maximal levels in luteal cells of pregnant rats where levels of PRL-R(s) approached those of PRL-R(L). The relative concentrations shifted from a 27-fold excess of PRL-R(L) in preovulatory granulosa cells to a 3.7-fold difference in luteal cells during midgestation. Despite the increased PRL-R(L) expression in differentiated granulosa cells, PRL did not stimulate detectable activation of Stats. Rather PRL activation of Stat5, principally Stat5b, occurred in association with luteinization. In contrast, granulosa cells of untreated immature and H rats contained a high level of DNA binding activity, which was shown to be comprised entirely of activated, phosphorylated Stat3. Treatment with estrogen and FSH reduced the amount of phosphorylated Stat3 and abolished its ability to bind DNA, an effect temporally related to increased PIAS-3. Expression of SHP-1 (but not SHP-2) was also hormonally regulated; SHP-1 mRNA and protein were high in granulosa cells of H rats, decreased by estrogen and FSH, and subsequently increased dramatically with luteinization. Of particular note, SHP-1 was localized in cytoplasm of granulosa cells in atretic follicles but was distinctly nuclear in luteal cells, indicative of different functional roles. Collectively, these results indicate that Stat3 and Stat5 are activated by distinct cytokine-signaling pathways modulated through differentiation-dependent transcriptional regulation of signaling pathway components and mediate distinct functional processes in the rat ovary: early follicle growth and atresia vs. luteinization.

X-Ray fluorescence imaging and other analyses identify selenium and GPX1 as important in female reproductive function.

Studies of selenium (Se) status indicate that Se is necessary for fertility but how precisely is not known. We aimed to show that Se was important in bovine female reproductive function. The elemental distribution in the bovine ovary (n = 45 sections) was identified by X-ray fluorescence (XRF) imaging. Se was consistently localized to the granulosa cell layer of large (>10 mm) healthy follicles. Inductively Coupled Plasma - Mass Spectrometry revealed tenfold higher Se in the bovine follicle wall compared to corpora lutea. Gene expression analysis of selenoprotein genes GPX1, GPX3, VIMP and SELM in bovine granulosa cells revealed that only GPX1 was significantly up-regulated in large healthy follicles compared to the small healthy or atretic follicles (P < 0.05). Western immunoblotting identified GPX1 protein in bovine granulosa cells of large healthy follicles, but not of small healthy follicles. To assess if GPX1 was important in human follicles, cumulus cells from women undergoing IVF/ICSI with single embryo transfer were collected. Oocytes and embryos were cultured and transferred independently in 30 patients undergoing elective single embryo transfer. Gene expression of GPX1 was significantly higher in human cumulus cells from cumulus-oocyte complexes yielding a pregnancy (P < 0.05). We present the first XRF imaging of mammalian ovaries showing that Se is consistently localized to the granulosa cells of large healthy follicles. We conclude that Se and selenoproteins are elevated in large healthy follicles and may play a critical role as an antioxidant during late follicular development.

Correction: X-Ray fluorescence imaging and other analyses identify selenium and GPX1 as important in female reproductive function.

Correction for 'X-Ray fluorescence imaging and other analyses identify selenium and GPX1 as important in female reproductive function' by M. J. Ceko et al., Metallomics, 2014, DOI: .

Immunization against the N-terminal peptide of the inhibin alpha 43-subunit (alpha N) disrupts tissue remodeling and the increase in matrix metalloproteinase-2 during ovulation.

Immunization of ewes against the N-terminal peptide of inhibin alpha 43 (alpha N) reduces fertility; this is thought to be due to impaired oocyte release at ovulation. This study further investigates the effect of alpha N immunoneutralization on the ovulatory process. Light microscopy was used to examine the effects of alpha N immunization of the tissue-remodeling process during ovulation and formation of the corpus luteum (CL) structure. Changes in follicular levels of matrix metalloproteinase-2 (MMP-2) with approaching ovulation were also investigated in normal and alpha N-immunized ewes. Differences in structure of 2-day-old CL were observed between control and alpha N-immunized ewes. Control CL had confluent luteal tissue throughout the internal structure and invaginations of theca and vasculature were common and penetrated deep into the luteal tissue. Immunized ewe CL had large fluid-filled antra, giving them a cystic appearance; luteal tissue remained a thin 10- to 15-cell layer lining the wall surrounding the antrum. Infolding of the surrounding tissue was incomplete, and thecal/vascular invaginations were rare and failed to penetrate into the luteal tissue. Morphologically normal rupture stigma were seen at the apex of both control and alpha N-immunized CL. Gelatin-digesting activity in follicular fluid collected 0, 12, and 24 h after hCG administration in control ewes increased significantly as the time of ovulation approached (827 +/- 182, 842 +/- 159, and 1230 +/- 89 mU/ml, respectively, in Exp 1; 743 +/- 32, 1182 +/- 98, and 1306 +/- 91 mU/ml at the same times in Exp 2). alpha N immunization reduced follicular gelatinase activity at each time in Exp 1 (533 +/- 132, 740 +/- 67, and 809 +/- 147 mU/ml) and Exp 2 (587 +/- 21, 768 +/- 27, and 891 +/- 53 mU/ml); the reduction was significant at 24 h in Exp 1 and at all times in Exp 2. Gelatin zymography of follicular fluid revealed bands of gelatinase of 72/67 kilodaltons, consistent with latent and active MMP-2. The area digested by both latent and active MMP-2 increased with approaching time of ovulation and was reduced by alpha N immunization. These data suggest that MMP-2 has a role in the tissue-remodeling processes of ovulation and CL formation in the ewe and that immunization against alpha N, which impairs fertility, effects the preovulatory cascade of intrafollicular proteolytic activity, reducing MMP-2 levels and disrupting normal CL formation.

Induction of early growth response protein-1 gene expression in the rat ovary in response to an ovulatory dose of human chorionic gonadotropin.

Granulosa cells in a mature ovarian follicle have an abundance of LH/hCG receptors that respond rapidly to an ovulatory surge in gonadotropins. Within minutes, membrane signal transduction sets in motion metabolic changes that lead to follicular rupture. This study provides evidence that the initial ovarian response to such an ovulatory stimulus includes induction of the immediate-early transcription factor gene for early growth response protein-1 (Egr-1). Immature Wistar rats were primed with 10 IU equine CG (eCG), sc, and 48 h later the 12-h ovulatory process was initiated by 10 IU hCG, sc. Ovarian RNA was extracted at 0, 0.5, 1, 2, 4, 8, 12, and 24 h after the primed animals were injected with hCG. The RNA extracts were used for RT-PCR differential display for random detection of gene expression in the stimulated ovarian tissue. Northern analysis of one of the differentially amplified complementary DNAs confirmed that it was part of a gene that was significantly up-regulated within 1 h after the ovaries had been stimulated by hCG. Maximum transcription was at 4 h after hCG, and expression declined to 0 h control levels by 24 h after hCG. Subcloning and sequence analysis revealed that the complementary DNA matched the gene for Egr-1. In situ hybridization indicated that the Egr-1 messenger RNA was in the granulosa layer of mature follicles. Western blotting confirmed the temporal pattern of Egr-1 expression detected by differential display, Northern analysis and in situ hybridization. The Egr-1 protein is approximately 84 kDa. In conclusion, the data show that expression of the zinc finger transcription factor Egr-1 is an early event in the cascade of inflammatory-like changes that occur in an ovulatory follicle in response to a trophic hormone.

Expression of tumor necrosis factor-stimulated gene-6 in the rat ovary in response to an ovulatory dose of gonadotropin.

Current evidence supports the hypothesis that the biochemical events of mammalian ovulation are analogous to an acute inflammatory reaction. This study reveals that tumor necrosis factor-stimulated gene-6 (TSG-6), which encodes a member of the superfamily of hyaluronan-binding proteins that is specifically translated in inflammatory reactions, is expressed in ovarian follicles that have been induced to ovulate. Immature Wistar rats were primed with 10 IU equine CG s.c.; and 48 h later, the 12-h ovulatory process was initiated by 10 IU human CG (hCG), s.c.. Ovarian RNA was extracted at 0, 2, 4, 8, 12, and 24 h after the primed animals were injected with hCG. The RNA extracts were used for RT-PCR differential display of amplified complementary DNAs (cDNAs) that represented gene expression in the stimulated ovarian tissue. Northern analysis of one of the differentially amplified cDNAs confirmed that it was part of a gene that was substantially up-regulated at 4-8 h after the ovaries had been stimulated by hCG. Subcloning and sequence analysis revealed that the cDNA matched the gene for TSG-6. In situ hybridization indicated that the TSG-6 messenger RNA was primarily located in the cumulus mass and the antral granulosa cells of large ovarian follicles. In conclusion, the data show that expression of TSG-6 is an integral part of the cascade of inflammatory-like changes that occur in an ovulatory follicle in response to a trophic hormone that couples with luteinizing hormone/hCG receptors.

Regulation of transcripts encoding ADAMTS-1 (a disintegrin and metalloproteinase with thrombospondin-like motifs-1) and progesterone receptor by human chorionic gonadotropin in equine preovulatory follicles.

One member of a new family of metalloproteinases, a disintegrin and metalloproteinase with thrombospondin-like motifs-1 (ADAMTS-1), has been found to be expressed and hormonally induced in granulosa cells of ovulating rodent follicles. Furthermore, the targeted disruption of the ADAMTS-1 gene resulted in ovarian defects associated with severely impaired fertility. While these data demonstrate the importance of ADAMTS-1 in rodent ovarian physiology, the potential role of ADAMTS-1 in the ovulatory process of monoovulatory species remains unknown. The objectives of this study were to clone the equine ADAMTS-1 primary transcript and to study its regulation during human chorionic gonadotropin (hCG)-induced ovulation. A 3573 bp follicular cDNA library clone was isolated and found to encode a nearly complete, highly conserved ADAMTS-1 homologue. Real-time RT-PCR analysis detected this transcript in diverse tIssues, including previously unreported sites of ADAMTS-1 expression such as the male reproductive tract, the follicular theca interna and the mature corpus luteum. The tIssue distribution of the progesterone receptor (PR), a known regulator of ADAMTS-1 expression in rodent preovulatory follicles, was found to overlap that of ADAMTS-1 in some tIssues. A study of the regulation of follicular ADAMTS-1 and PR mRNAs during the hCG-induced ovulatory process revealed distinct patterns of regulation in granulosa cells and in theca interna. In granulosa cells, ADAMTS-1 mRNA was found to be induced at 12 h post-hCG (P<0.05), followed by a return to basal levels by 30 h and a re-increase at 33-39 h (P<0.05). A concomitant increase in PR mRNA (P<0.05) was observed at 12 h post-hCG. In theca interna, abundant ADAMTS-1 mRNA was detected at all timepoints, and levels increased transiently at 33 h post-hCG (P<0.05), whereas no significant change was observed in PR mRNA. Together, these data demonstrate for the first time the hormonally regulated ovarian expression of ADAMTS-1 in a monoovulatory species, and identify a novel biphasic regulation of ADAMTS-1 in granulosa cells and a regulated expression in theca interna that were not previously observed in rodents.

Glycosylation of purified enveloped nucleocapsids of the granulosis virus infecting Plodia interpunctella as determined by lectin binding.

Using various iodinated plant lectins and a sensitive in vitro lectin binding procedure in which the radiolabeled lectins are applied directly to viral proteins on nitrocellulose sheets transferred from sodium dodecyl sulfate-polyacrylamide gels (Bartles and Hubbard, 1984; Glass et al., 1981), the proteins of the granulosis virus infecting Plodia interpunctella were probed for carbohydrate moieties. Six proteins (Mr 39 700, 31 000, 29 900, 26 300, 17 800 and 12 600) could be detected using a probe for alpha-D-N-acetylgalactosamine. Three proteins were also detectable with the probe specific for alpha-L-fucose (Mr 44 900, 31 000 and 17 800). One protein (Mr 17 800) was detected with the probe for sialic acid.