Nancy G. Pedigo

The role of ovarian proteases and their inhibitors in ovulation

To assess the role of inhibitors of proteolytic enzymes, such as plasminogen activator (PA) and collagenase in the ovulatory process, inhibitor activity and mRNA levels were examined in periovulatory rat and human ovaries. In the rat, immature animals received 20 IU of pregnant mare serum gonadotropin (PMSG) followed 52 h later by 10 IU of hCG. Ovaries were removed at intervals from 0 to 20 h after human chorionic gonadotropin (hCG) administration. Inhibitor activity for metalloproteinases, such as collagenase, increased from 60.5 +/- 4.1 inhibitor units/ovary at 0 h (i.e., time of hCG treatment) to a maximum of 218.2 +/- 11.4 units/ovary at 8 h after hCG before decreasing at 12 h (time of ovulation) and 20 h (122.2 +/- 7.9 and 71.6 +/- 8.1 units/ovary, respectively). Human follicular fluid and granulosa cells were obtained from preovulatory follicles of patients in our in vitro fertilization program. Metalloproteinase inhibitor activity was evaluated in follicular fluid as well as the levels of PA and PA inhibitor (PAI) mRNA by Northern analysis. Increasing metalloproteinase inhibitor activity was positively correlated with follicular levels of estradiol (p less than 0.001) and progesterone (p less than 0.02, N = 26). Chromatographic separation of follicular fluid resulted in two peaks of metalloproteinase inhibitor activity. The large molecular weight (MW) inhibitor had an approximate size of 700 kilodaltons (kDa) and may represent alpha 2-macroglobulin, a serum-derived inhibitor. The small MW inhibitor shared many of the characteristics of tissue-derived inhibitors of metalloproteinases. Partial purification of the small MW inhibitor by Concanavalin A-Sepharose and Heparin-Sepharose chromatography demonstrated the inhibitor to be a glycoprotein with an approximate MW = 28-29 K. Northern analysis of human granulosa cell total RNA from preovulatory follicles showed little or no detectable tissue-type PA or urokinase-type PA mRNA. In contrast, two species of PA inhibitor type-1 mRNA were detected in relative abundance. The present findings demonstrate the presence of proteolytic inhibitors in periovulatory ovaries of the rat and human. These ovarian inhibitors may play a role in regulating connective tissue remodeling during follicular rupture.

Characterization of a computerized semen analysis system

In the present studies we have evaluated the optimal operating conditions for the Hamilton-Thorn HTM-2000 computerized semen analyzer (Hamilton-Thorn, Danvers, MA). The best reproducibility in measurement of sperm concentration was obtained using 20 frames acquired at 19 frames/s. The measurement of sperm concentration was not adversely affected by the number of fields analyzed. The intrasample and intersample coefficients of variation for sperm concentration were 9.5% and 25.5%; sperm motility, 18.4% and 28.9%; lateral head displacement, 16.5% and 19.9%; path velocity, 6.8% and 13.9%; progressive velocity, 4.5% and 9.9%; and linear index, 2.5% and 4.2%; respectively. These differences suggest that sampling error has a significant influence on the reliability of sperm evaluation. The precision and rapidity of the HTM-2000 compares favorably with data previously reported from other systems available for clinical semen analysis.

Embryonic losses after 10 week administration of cobalt to male mice

Cobalt toxicity was evaluated in the dominant lethal assay (DLA) to determine whether the detrimental effects of cobalt on spermatozoa would have an impact on offspring. Male B6C3F1 mice were treated with cobaltous chloride (400 ppm Co) for 10 weeks and mated. Neither the stage nor rate of development in vitro of 2-cell embryos to blastocyst from cobalt-treated males was affected. There was an increase in preimplantation losses and a decrease in total and live births, but no change in postimplantation losses from litters at day 19 of gestation. Fertility of the males was maintained during the 10-week cobalt treatment period, decreased during the DLA, and recovered over the next 6 weeks. Sperm parameters at the end of DLA and the recovery period showed that cobalt decreased all parameters measured at 12 weeks, but these parameters, except concentration, recovered to control levels by 18 weeks. Tissue concentrations of cobalt measured by atomic absorption analysis were increased in liver, kidney, testis, and epididymis after 12 weeks of cobalt treatment. We conclude that cobalt affected preimplantation losses in the DLA by compromising the fertility of treated males.

A 5′-Distal Element Mediates Vitamin D-Inducibility of PDGF-A Gene Transcription

Expression of the platelet-derived growth factor-A subunit (PDGF-A) is regulated to a significant degree by DNA elements located in the 5′-distal region of the gene. A potent basal enhancer (ACE66) located approximately 7 kb upstream of the transcription start site contains a number of half-sites for nuclear receptor binding, two of which are arranged in the form of direct repeat-3 motif that corresponds to a consensus vitamin D response element (VDRE). Electrophoretic mobility shift assays confirmed that the ACE66 sequence was recognized as a high affinity target for binding of heterodimers of recombinant vitamin D receptor (VDR) and its partner, retinoid-X receptor-α (RXRα). VDRE activity was localized by transient transfection analysis to a direct repeat-3 motif within the 5′-portion of the ACE66 element. Moreover, 1,25-(OH)2D3 was validated as a regulator of the endogenous PDGF-A gene by the vitamin D-stimulated upregulation of PDGF-A mRNA levels in a VDR-expressing clone of JEG-3 cells. Thus, PDGF-A represents a novel mitogenic target of 1,25-(OH)2D3 whose expression is induced via binding of hormone-activated VDR to a response element located far upstream of the transcription start site.

A dominant-negative mutant of the platelet-derived growth factor A-chain increases survival of hamsters implanted intracerebrally with the highly invasive CxT24-neo3 glioblastoma cell

Evidence is accumulating to suggest a role for PDGF in stimulating malignant growth in astrocytoma, although it has been obtained using model systems (growth in 2-dimensional cell culture, athymic nude mice) that do not assess the complex interactions of these tumors with normal brain tissue. In the current study, the highly invasive hamster glioblastoma cell line CxT24-neo3 was used as a model to study the role of platelet-derived growth factor (PDGF) in mediating malignant growth both in vitro and in vivo when implanted directly into the right lateral ventricle of the brain. Co-expression of PDGF B-chain mRNA and PDGF α-receptors was detected in these cells, indicating potential for autocrine activation of their growth. CxT24-neo3 cells transfected with wild-type and receptor binding-deficient forms of the PDGF A- and B-chains displayed alterations in their abilities to grow as three-dimensional spheroids, with overexpression of wild-type B-chain resulting in increased spheroid formation, but a decreased rate of spheroid growth. Influence of these PDGF polypeptides on tumor invasion and survival time in vivo was evaluated following implantation of these spheroids in the brain. While all hamsters implanted with control spheroids died within 21 d (average 17 d), those implanted with cells expressing the receptor binding-deficient A-chain survived for much greater periods of time (average 80 d). Modest increases in survival were also seen in cells stably expressing wild-type A-chain (25 d) and mutant B-chain (26 d) proteins. The present study suggests an important role of PDGF in mediating the malignant growth of the CxT24-neo3 cell line in cerebral cortex, possibly via paracrine interactions with normal cortical cell types (i.e., glia, neurons).

The effects of cyclic adenosine monophosphate, forskolin, and theophylline on motility parameters in gossypol-treated human sperm

OBJECTIVE To examine the effect of gossypol on human sperm in vitro and the mechanism for the effect. DESIGN Fresh sperm ejaculates obtained from normal donors to the University of Kentucky Andrology Donor Program were exposed to gossypol. Motility was studied manually and using computer-assisted sperm analysis. In subsequent experiments, the effects of forskolin, theophylline, and cyclic adenosine monophosphate (cAMP) on sperm motion were measured. SETTING University of Kentucky Department of Obstetrics and Gynecology Andrology Laboratory. MAIN OUTCOME MEASURES Manual and computer-assisted measurements of sperm motility and motion characteristics. RESULTS Gossypol inhibited sperm motility, which could be reversed partially by increasing cAMP. CONCLUSION Gossypol exposure in vitro adversely affects sperm motility in a dose- and time-dependent manner by a cAMP-dependent mechanism.

THE CALCITRIOL ANALOGUE EB1089 IMPAIRS ALVEOLARIZATION AND INDUCES LOCALIZED REGIONS OF INCREASED FIBROBLAST DENSITY IN NEONATAL RAT LUNG

The active form of vitamin D3, 1,25-dihydroxyvitamin D3 (1,25-(OH)2D3, or calcitriol), is a potent mitogen for fibroblasts cultured from rat lungs at postnatal day 4 (P4), during the peak of septation (P3 to P7). In light of the key role of fibroblasts in alveolar septation, the authors conducted studies to measure the extent to which 1,25-(OH)2D3 affects lung maturation in vivo, as well as its ability to influence the stimulatory activity of all-trans retinoic acid (RA). To identify a calcitriol analogue with maximal mitogenic activity and low systemic toxicity, two compounds with reduced calcemic activity (EB1089 and CB1093) and a superagonist (MC1288) were evaluated in neonatal rat lung fibroblast cultures. All 3 analogues were more potent mitogens than 1,25-(OH)2D3 itself (MC1288 ∼ CB1093 > EB1089 > 1,25-(OH)2D3). In addition, each was more effective than 1,25-(OH)2D3(EB1089 > CB1093 > MC1288 > 1,25-(OH)2D3) in the activation of a vitamin D response element from the platelet-derived growth factor (PDGF)-A gene, whose expression is essential for normal alveolarization. Daily administration of EB1089 to rats 4 to 12 days of age caused an increase in mean alveolar chord length (P <.0001), and also elicited prominent regions of fibroblast hypercellularity, as defined in terms of a vimentin-positive, factor VIII–negative phenotype. EB1089 and RA each induced the expression of 2 important lung structural proteins, collagen and elastin. Regions of fibroblast hypercellularity induced by EB1089 were strongly positive for expression of the alveolarization-relevant growth factors, PDGF-AA and vascular endothelial growth factor (VEGF). These studies demonstrate that 1,25-(OH)2D3 disrupts the overall alveolarization process in the neonatal lung, although it stimulates expression of some proteins associated with lung morphogenesis.

A 5'-distal enhanceosome in the PDGF-A gene is activated in choriocarcinoma cells via ligand-independent binding of vitamin D receptor and constitutive jun kinase signaling.

Overexpression of platelet-derived growth factor A-chain (PDGF-A) is clearly linked to autocrine and paracrine stimulation of malignant growth in many human cancers. We have shown previously that PDGF-A overexpression in choriocarcinoma, hepatoma and lung carcinoma cell lines is driven by the activity of a 66 bp enhancer element (ACE66) located approximately 7 kb upstream of the PDGF-A transcription start site. In this study, the ACE66 element is shown to be activated in JEG-3 choriocarcinoma cells through synergistic interactions between consensus DNA motifs for binding of vitamin D receptor, AP1 and ELK1. Binding of the vitamin D/retinoid-X receptor (VDR/RXRα) heterodimer to the ACE66 element was reconstituted in vitro with recombinant VDR/RXRα and with JEG-3 nuclear extract, and was verified in living JEG-3 cells by chromatin immunoprecipitation analysis. Transcriptional activity of the ACE66 element, as well as occupancy of the element by VDR/RXRα, was shown to be independent of stimulation with the hormonal VDR ligand, 1,25-dihydroxyvitamin D3. The jun kinase pathway of mitogen-activated protein kinase (MAPK) signaling was shown to activate the ACE66 enhancer, most likely through activation of factors binding to the AP1 element. These results identify a novel mechanism of transcriptional enhancement involving ligand-independent activity of the VDR/RXR heterodimer and MAPK signaling pathways that appears to play an important role in the overexpression of PDGF in many different settings of human malignancy.