Chemokine Ligand 20: A Signal for Leukocyte Recruitment During Human Ovulation?

Ovulation is one of the cornerstones of female fertility. Disruption of the ovulatory process results in infertility, which affects approximately 10% of couples. Using a unique model in which the dominant follicle is collected across the periovulatory period in women, we have identified a leukocyte chemoattractant, chemokine ligand 20 (CCL20), in the human ovary. CCL20 mRNA is massively induced after an in vivo human chorionic gonadotropin (hCG) stimulus in granulosa (>10 000-fold) and theca (>4000-fold) cells collected during the early ovulatory (12-18 h) and late ovulatory (18-34 h) periods after hCG administration. Because the LH surge sets in motion an inflammatory reaction characterized by an influx of leukocytes and CCL20 is known to recruit leukocytes in other systems, the composition of ovarian leukocytes (CD45+) containing the CCL20 receptor CCR6 was determined immediately prior to ovulation. CD45+/CCR6+ cells were primarily natural killer cells (41%) along with B cells (12%), T cells (11%), neutrophils (10%), and monocytes (9%). Importantly, exogenous CCL20 stimulated ovarian leukocyte migration 59% within 90 minutes. Due to the difficulties in obtaining human follicles, an in vitro model was developed using granulosa-lutein cells to explore CCL20 regulation. CCL20 expression increased 40-fold within 6 hours after hCG, was regulated partially by the epithelial growth factor pathway, and was positively correlated with progesterone production. These results demonstrate that hCG dramatically increases CCL20 expression in the human ovary, that ovarian leukocytes contain the CCL20 receptor, and that CCL20 stimulates leukocyte migration. Our findings raise the prospect that CCL20 may aid in the final ovulatory events and contribute to fertility in women.

wrwyrggrywrw is a single-chain functional analog of the Holliday junction-binding homodimer, (wrwycr)2.

DNA repair pathways in bacteria that use homologous recombination involve the formation and subsequent resolution of Holliday junction (HJ) intermediates. We have previously identified several hexameric peptides that bind to HJs and interfere with HJ processing enzymes in vitro. The peptide WRWYCR and its D-amino acid stereoisomer wrwycr, are potent antibacterial agents. These hexapeptides must form homodimers in order to interact stably with HJs, and inhibit bacterial growth, and this represents a potential limitation. Herein we describe a disulfide bond-independent inhibitor, WRWYRGGRYWRW and its D-stereoisomer wrwyrggrywrw. We have characterized these single-chain, linear analogs of the hexapeptides, and show that in addition to effectively binding to HJs, and inhibiting the activity of DNA repair enzymes that process HJs, they have equal or greater potency against Gram-positive and Gram-negative bacterial growth. The analogs were also shown to cause DNA damage in bacteria, and disrupt the integrity of the bacterial cytoplasmic membrane. Finally, we found that they have little toxicity toward several eukaryotic cell types at concentrations needed to inhibit bacterial growth.

Potent antimicrobial small molecules screened as inhibitors of tyrosine recombinases and Holliday junction-resolving enzymes.

Holliday junctions (HJs) are critical intermediates in many recombination-dependent DNA repair pathways. Our lab has previously identified several hexameric peptides that target HJ intermediates formed in DNA recombination reactions. One of the most potent peptides, WRWYCR, is active as a homodimer and has shown bactericidal activity partly because of its ability to interfere with DNA repair proteins that act upon HJs. To increase the possibility of developing a therapeutic targeting DNA repair, we searched for small molecule inhibitors that were functional surrogates of the peptides. Initial screens of heterocyclic small molecule libraries resulted in the identification of several N-methyl aminocyclic thiourea inhibitors. Like the peptides, these inhibitors trapped HJs formed during recombination reactions in vitro, but were less potent than the peptides in biochemical assays and had little antibacterial activity. In this study, we describe the screening of a second set of libraries containing somewhat larger and more symmetrical scaffolds in an effort to mimic the symmetry of a WRWYCR homodimer and its target. From this screen, we identified several pyrrolidine bis-cyclic guanidine inhibitors that also interfere with processing of HJs in vitro and are potent inhibitors of Gram-negative and especially Gram-positive bacterial growth. These molecules are proof-of-principle of a class of compounds with novel activities, which may in the future be developed into a new class of antibiotics that will expand the available choices for therapy against drug-resistant bacteria.

Current results with slow freezing and vitrification of the human oocyte.

The past decade has witnessed renewed interest in human oocyte cryopreservation (OCP). This article reviews the two general methods used for OCP, slow freezing and vitrification, compares the outcomes associated with each technique and discusses the factors that might influence success with OCP (such as oocyte selection or day of transfer). Based on available data, OCP offers a reliable, reproducible method for preservation of the female gamete and will find increasing application in assisted reproductive technology. Oocyte cryopreservation can provide a number of advantages to couples undergoing assisted reproduction or to women interested in fertility preservation. Two methods, slow freezing and vitrification, have been used successfully for oocyte cryopreservation. This article reviews and compares these methods, and discusses various factors that can impact upon success of oocyte cryopreservation.

Oocyte cryopreservation: is it time to remove its experimental label?

As more reproductive-age women survive cancer at the expense of gonadotoxic therapy, the need for viable fertility preservation options has become paramount. Embryo cryopreservation, often using donor sperm, has been the standard offered these women over the past 20 years. Preservation of unfertilized oocytes now represents an acceptable and often equally viable alternative, particularly for single women, due to technologic advances made in the past decade. Given such, oocyte cryopreservation's experimental designation and need for IRB approval should thus be revisited.

Peptide wrwycr inhibits the excision of several prophages and traps holliday junctions inside bacteria.

Peptide inhibitors of phage lambda site-specific recombination were previously isolated by screening synthetic combinatorial peptide libraries. These inhibitors cause the accumulation of complexes between the recombinase and the Holliday junction intermediate of several highly divergent tyrosine recombinases. Peptide WRWYCR and its d-amino acid derivative bind to the center of protein-free junctions and prevent their resolution either by site-specific recombinases or by junction resolvases or helicases. With lesser affinity, the peptides also bind to branched DNA molecules that mimic replication forks. The peptides are bactericidal to both gram-positive and gram-negative bacteria, presumably because they can interfere with DNA repair and with chromosome dimer resolution by the XerC and XerD tyrosine recombinases. In order to test the correspondence between their mechanism in vivo and in vitro, we have tested and shown peptide wrwycr's ability to inhibit the excision of several prophages (lambda, P22, Gifsy-1, Gifsy-2, Fels-1, Fels-2) and to trap Holliday junction intermediates of phage lambda site-specific recombination in vivo. In addition, we found that the peptide inhibits replication of the Salmonella prophage Fels-1 while integrated in the chromosome. These findings further support the proposed mechanistic basis for the antimicrobial activity of the peptide and its use as a tool to dissect strand exchange-dependent DNA repair within cells.

Initial experience with a donor egg bank.

OBJECTIVE: To report on the establishment of a commercial donor egg bank (CryoEggs International, LP) and to present our initial experience from the first four patients to receive eggs. DESIGN: Case report. SETTING: Private fertility clinic. PATIENT(S): The four recipient women were aged 43, 43, 40, and 33 years. All had cycle day FSH levels greater than 25 mIU/mL. All were given the option of fresh donor egg IVF but opted to use frozen donor oocytes. INTERVENTION(S): Purchased and quarantined frozen donor eggs were thawed and inseminated using intracytoplasmic sperm injection (ICSI). Subsequent embryos were transferred on day 3. MAIN OUTCOME MEASURE(S): Clinical pregnancy as defined by presence of cardiac activity. RESULT(S): There was a thawed egg survival rate of 76%, a fertilization rate of 74%, a pregnancy rate (PR) of 50%, with an average of 2.75 embryos per transfer and an implantation rate of 27%. CONCLUSION(S): Although very preliminary, these results indicate that more widespread use of frozen donor eggs obtained from a commercial egg bank may be feasible in the future, changing the landscape of donor egg IVF.

Spermidine biases the resolution of Holliday junctions by phage lambda integrase.

Holliday junctions are a central intermediate in diverse pathways of DNA repair and recombination. The isomerization of a junction determines the directionality of the recombination event. Previous studies have shown that the identity of the central sequence of the junction may favor one of the two isomers, in turn controlling the direction of the pathway. Here we demonstrate that, in the absence of DNA sequence-mediated isomer preference, polycations are the major contributor to biasing strand cleavage during junction resolution. In the case of wild-type phage lambda excision junctions, spermidine plays the dominant role in controlling the isomerization state of the junction and increases the rate of junction resolution. Spermidine also counteracts the sequence-imposed bias on resolution. The spermidine-induced bias is seen equally on supercoiled and linear excisive recombination junction intermediates, and thus is not just an artefact of in vitro recombination conditions. The contribution of spermidine requires the presence of accessory factors, and results in the repositioning of Int's core-binding domains on junctions, perhaps due to DNA-spermidine-protein interactions, or by influencing DNA conformation in the core region. Our results lead us to propose that spermidine together with accessory factors promotes the formation of the second junction isomer. We propose that this rearrangement triggers the activation of the second pair of Int active sites necessary to resolve Holliday junctions during phage lambda Int-mediated recombination.

Human oocyte cryopreservation: 5-year experience with a sodium-depleted slow freezing method.

A slow freezing/rapid thawing method for the cryopreservation of human oocytes has been employed using a sodium-depleted culture media. In 53 frozen egg-embryo transfer (FEET) cycles, a 60.4% survival rate post-thaw was obtained and a 62.0% fertilization rate following intracytoplasmic sperm injection. Overall pregnancy rates were 26.4% per thaw attempt, 30.4% per patient, and 32.6% per embryo transfer. Pregnancy rates using sodium-depleted phosphate-buffered saline (PBS) as the base medium were 20.0% per thaw, 21.7% per patient, and 26.3% per transfer. With sodium-depleted modified human tubal fluid (mHTF) as the base for the cryopreservation medium, rates were 32.1% per thaw attempt, 39.1% per patient, 37.5% per transfer. The overall implantation rates were 4.2% per thawed oocyte and 13.6% per embryo, (PBS: 3.0% per egg, 10.6% per embryo; mHTF:5.3% per oocyte; 15.9% per embryo). These data indicate that the use of a sodium-depleted media with slow freezing and rapid thawing can yield acceptable pregnancy rates after FEET.

Holliday junction-binding peptides inhibit distinct junction-processing enzymes.

Holliday junctions (HJ) are the central intermediates in both homologous recombination and site-specific recombination performed by tyrosine recombinases such as the bacteriophage lambda Integrase (Int) protein. Previously, our lab identified peptide inhibitors of Int-mediated recombination that prevent the resolution of HJ intermediates. We now show that two of these inhibitors bind HJ DNA in the square-planar conformation even in the absence of Int protein. The peptides prevent unwinding of branched DNA substrates by the RecG helicase of Escherichia coli and interfere with the resolution of HJ substrates by the RuvABC complex. Our results suggest that these peptides target all proteins that process HJ in the square-planar conformation. These inhibitors should be extremely useful for dissecting homologous recombination and recombination-dependent repair in vitro and in vivo.

Reversible inhibitors of lambda integrase-mediated recombination efficiently trap Holliday junction intermediates and form the basis of a novel assay for junction resolution.

The bacteriophage lambda integrase catalyzes four site-specific recombination pathways with distinct protein and DNA requirements and nucleoprotein intermediates. Some of these intermediates are very transient and difficult to obtain in significant amounts, due to the high efficiency and processivity of integrase, the lack of requirements for external energy factors or metal ions, and the highly reversible nature of each of the intermediates. We have previously used mixture-based combinatorial libraries to identify hexapeptides that trap 40-60% of recombination substrates at the Holliday junction stage of the reaction. These inhibitors discriminate between the four pathways, blocking one of them (bent-L recombination) more severely than the others and blocking the excision pathway least. We presume that these differences reflect specific conformational differences of the nucleoprotein intermediates in each pathway. We have now identified new inhibitors of the excision pathway. One of these, WRWYCR, is over 50-fold more potent at inhibiting excision than the previously identified peptides. This peptide stably traps Holliday junction complexes in all recombination pathways mediated by integrase as well as Cre. This finding and other data presented indicate that the peptide's target is a common feature shared by the Holliday junction complexes assembled by tyrosine recombinases. We have taken advantage of reversible inhibition by the active peptides to develop a new assay for Holliday junction resolution. This assay is particularly useful for determining junction resolution rates in cases where complexes directly assembled on junction substrates undergo little or no catalysis.

Human oocyte cryopreservation as an adjunct to IVF-embryo transfer cycles.

BACKGROUND: The purpose of this work was to develop methods for successful cryopreservation of human oocytes. METHODS: Two cryopreservation procedures were used. Method 1 involved use of 1.5 mol/l propanediol (PrOH)-0.1 mol/l sucrose with medium containing sodium (Na) as cryoprotectant medium, seeding at -7 degrees C, and stepwise dilution of cryoprotectant post-thaw. Method 2 used Na-depleted media with 1.5 mol/l PrOH-0.2 mol/l sucrose for freezing, seeding at -6 degrees C, and use of high sucrose (0.5 and 0.2 mol/l) for cryoprotectant removal. RESULTS: The first method was used in seven patients, and gave poor (12.3%) survival results and no pregnancies. The second method was used in 15 patients (16 cycles), and yielded good survival and fertilization rates (74.4 and 59% respectively), with four pregnancies and five healthy infants born to 11 women receiving an embryo transfer. CONCLUSIONS: Using Na-depleted media along with other alterations in freezing and thawing procedures, human oocyte cryopreservation can provide excellent survival and pregnancy rates.

Mutations at residues 282, 286, and 293 of phage lambda integrase exert pathway-specific effects on synapsis and catalysis in recombination.

Bacteriophage lambda integrase (Int) catalyzes site-specific recombination between pairs of attachment (att) sites. The att sites contain weak Int-binding sites called core-type sites that are separated by a 7-bp overlap region, where cleavage and strand exchange occur. We have characterized a number of mutant Int proteins with substitutions at positions S282 (S282A, S282F, and S282T), S286 (S286A, S286L, and S286T), and R293 (R293E, R293K, and R293Q). We investigated the core- and arm-binding properties and cooperativity of the mutant proteins, their ability to catalyze cleavage, and their ability to form and resolve Holliday junctions. Our kinetic analyses have identified synapsis as the rate-limiting step in excisive recombination. The IntS282 and IntS286 mutants show defects in synapsis in the bent-L and excisive pathways, respectively, while the IntR293 mutants exhibit synapsis defects in both the excision and bent-L pathways. The results of our study support earlier findings that the catalytic domain also serves a role in binding to core-type sites, that the core contacts made by this domain are important for both synapsis and catalysis, and that Int contacts core-type sites differently among the four recombination pathways. We speculate that these residues are important for the proper positioning of the catalytic residues involved in the recombination reaction and that their positions differ in the distinct nucleoprotein architectures formed during each pathway. Finally, we found that not all catalytic events in excision follow synapsis: the attL site probably undergoes several rounds of cleavage and ligation before it synapses and exchanges DNA with attR.