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Replication protein A (RPA) is a eukaryotic single-stranded (ss) DNA-binding (SSB) protein that is essential for all aspects of genome maintenance. RPA binds ssDNA with high affinity but can also diffuse along ssDNA. By itself, RPA is capable of transiently disrupting short regions of duplex DNA by diffusing from a ssDNA that flanks the duplex DNA. Using single-molecule total internal reflection fluorescence and optical trapping combined with fluorescence approaches, we show that S. cerevisiae Pif1 can use its ATP-dependent 5' to 3' translocase activity to chemomechanically push a single human RPA (hRPA) heterotrimer directionally along ssDNA at rates comparable to those of Pif1 translocation alone. We further show that using its translocation activity, Pif1 can push hRPA from a ssDNA loading site into a duplex DNA causing stable disruption of at least 9 bp of duplex DNA. These results highlight the dynamic nature of hRPA enabling it to be readily reorganized even when bound tightly to ssDNA and demonstrate a mechanism by which directional DNA unwinding can be achieved through the combined action of a ssDNA translocase that pushes an SSB protein. These results highlight the two basic requirements for any processive DNA helicase: transient DNA base pair melting (supplied by hRPA) and ATP-dependent directional ssDNA translocation (supplied by Pif1) and that these functions can be unlinked by using two separate proteins.

Escherichia coli UvrD DNA helicase functions in several DNA repair processes. As a monomer, UvrD can translocate rapidly and processively along ssDNA; however, the monomer is a poor helicase. To unwind duplex DNA in vitro, UvrD needs to be activated either by self-assembly to form a dimer or by interaction with an accessory protein. However, the mechanism of activation is not understood. UvrD can exist in multiple conformations associated with the rotational conformational state of its 2B subdomain, and its helicase activity has been correlated with a closed 2B conformation. Using single-molecule total internal reflection fluorescence microscopy, we examined the rotational conformational states of the 2B subdomain of fluorescently labeled UvrD and their rates of interconversion. We find that the 2B subdomain of the UvrD monomer can rotate between an open and closed conformation as well as two highly populated intermediate states. The binding of a DNA substrate shifts the 2B conformation of a labeled UvrD monomer to a more open state that shows no helicase activity. The binding of a second unlabeled UvrD shifts the 2B conformation of the labeled UvrD to a more closed state resulting in activation of helicase activity. Binding of a monomer of the structurally similar Escherichia coli Rep helicase does not elicit this effect. This indicates that the helicase activity of a UvrD dimer is promoted via direct interactions between UvrD subunits that affect the rotational conformational state of its 2B subdomain., Competing Interests: The authors declare no conflict of interest.

Escherichia coli RecBCD is a DNA helicase/nuclease that functions in double-stranded DNA break repair. RecBCD possesses two motors (RecB, a 3' to 5' translocase, and RecD, a 5' to 3' translocase). Current DNA unwinding models propose that motor translocation is tightly coupled to base pair melting. However, some biochemical evidence suggests that DNA melting of multiple base pairs may occur separately from single-stranded DNA translocation. To test this hypothesis, we designed DNA substrates containing reverse backbone polarity linkages that prevent ssDNA translocation of the canonical RecB and RecD motors. Surprisingly, we find that RecBCD can processively unwind DNA for at least 80bp beyond the reverse polarity linkages. This ability requires an ATPase active RecB motor, the RecB "arm" domain, and also the RecB nuclease domain, but not its nuclease activity. These results indicate that RecBCD can unwind duplex DNA processively in the absence of ssDNA translocation by the canonical motors and that the nuclease domain regulates the helicase activity of RecBCD., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

Notch signaling, involved in development and tissue homeostasis, is activated at the cell-cell interface through ligand-receptor interactions. Previous studies have implicated mechanical forces in the activation of Notch receptor upon binding to its ligand. Here we aimed to determine the single molecular force required for Notch activation by developing a novel low tension gauge tether (LTGT). LTGT utilizes the low unbinding force between single-stranded DNA (ssDNA) and Escherichia coli ssDNA binding protein (SSB) (∼4 pN dissociation force at 500 nm/s pulling rate). The ssDNA wraps around SSB and, upon application of force, unspools from SSB, much like the unspooling of a yoyo. One end of this nano yoyo is attached to the surface though SSB, while the other end presents a ligand. A Notch receptor, upon binding to its ligand, is believed to undergo force-induced conformational changes required for activating downstream signaling. If the required force for such activation is larger than 4 pN, ssDNA will unspool from SSB, and downstream signaling will not be activated. Using these LTGTs, in combination with the previously reported TGTs that rupture double-stranded DNA at defined forces, we demonstrate that Notch activation requires forces between 4 and 12 pN, assuming an in vivo loading rate of 60 pN/s. Taken together, our study provides a direct link between single-molecular forces and Notch activation.

Single-stranded (ss)DNA binding (SSB) proteins bind with high affinity to ssDNA generated during DNA replication, recombination, and repair; however, these SSBs must eventually be displaced from or reorganized along the ssDNA. One potential mechanism for reorganization is for an ssDNA translocase (ATP-dependent motor) to push the SSB along ssDNA. Here we use single molecule total internal reflection fluorescence microscopy to detect such pushing events. When Cy5-labeled Escherichia coli (Ec) SSB is bound to surface-immobilized 3'-Cy3-labeled ssDNA, a fluctuating FRET signal is observed, consistent with random diffusion of SSB along the ssDNA. Addition of Saccharomyces cerevisiae Pif1, a 5' to 3' ssDNA translocase, results in the appearance of isolated, irregularly spaced saw-tooth FRET spikes only in the presence of ATP. These FRET spikes result from translocase-induced directional (5' to 3') pushing of the SSB toward the 3' ssDNA end, followed by displacement of the SSB from the DNA end. Similar ATP-dependent pushing events, but in the opposite (3' to 5') direction, are observed with EcRep and EcUvrD (both 3' to 5' ssDNA translocases). Simulations indicate that these events reflect active pushing by the translocase. The ability of translocases to chemo-mechanically push heterologous SSB proteins along ssDNA provides a potential mechanism for reorganization and clearance of tightly bound SSBs from ssDNA.

The interferon-inducible, double-stranded (ds) RNA-activated protein kinase (PKR) contains a dsRNA-binding domain (dsRBD) and plays key roles in viral pathogenesis and innate immunity. Activation of PKR is typically mediated by long dsRNA, and regulation of PKR is disfavored by most RNA imperfections, including bulges and internal loops. Herein, we combine isothermal titration calorimetry (ITC), electrophoretic mobility shift assays, and small-angle X-ray scattering (SAXS) to dissect the thermodynamic basis for the specificity of the dsRBD termed "p20" for various RNAs and to detect any RNA conformational changes induced upon protein binding. We monitor binding of p20 to chimeric duplexes containing terminal RNA-DNA hybrid segments and a central dsRNA segment, which was either unbulged ("perfect") or bulged. The ITC data reveal strong binding of p20 to the perfect duplex (K(d) ~ 30 nM) and weaker binding to the bulged duplex (K(d) ~ 2-5 μM). SAXS reconstructions and p(r) distance distribution functions further uncover that p20 induces no significant conformational change in perfect dsRNA but largely straightens bulged dsRNA. Together, these observations support the dsRBD's ability to tightly bind to only A-form RNA and suggest that in a noninfected cell, PKR may be buffered via weak interactions with various bulged and looped RNAs, which it may straighten. This work suggests that PKR-regulating RNAs with complex secondary and tertiary structures likely mimic dsRNA and/or engage portions of PKR outside of the dsRBD.

The protein kinase PKR is activated by RNA to phosphorylate eIF-2α, inhibiting translation initiation. Long dsRNA activates PKR via interactions with the dsRNA-binding domain (dsRBD). Weakly structured RNA also activates PKR and does so in a 5'-triphosphate (ppp)-dependent fashion, however relatively little is known about this pathway. We used a mutant T7 RNA polymerase to incorporate all four triphosphate-containing nucleotides into the first position of a largely single-stranded RNA and found absence of selectivity, in that all four transcripts activate PKR. Recognition of 5'-triphosphate, but not the nucleobase at the 5'-most position, makes this RNA-mediated innate immune response sensitive to a broad array of viruses. PKR was neither activated in the presence of γ-GTP nor recognized NTPs other than ATP in activation competition and ITC binding assays. This indicates that the binding site for ATP is selective, which contrasts with the site for the 5' end of ppp-ssRNA. Activation experiments reveal that short dsRNAs compete with 5'-triphosphate RNAs and heparin for activation, and likewise gel-shift assays reveal that activating 5'-triphosphate RNAs and heparin compete with short dsRNAs for binding to PKR's dsRBD. The dsRBD thus plays a critical role in the activation of PKR by ppp-ssRNA and even heparin. At the same time, cross-linking experiments indicate that ppp-ssRNA interacts with PKR outside of the dsRBD as well. Overall, 5'-triphosphate-containing, weakly structured RNAs activate PKR via interactions with both the dsRBD and a distinct triphosphate binding site that lacks 5'-nucleobase specificity, allowing the innate immune response to provide broad-spectrum protection from pathogens.

The malachite green aptamer binds two closely related ligands, malachite green (MG) and tetramethylrosamine (TMR), with nearly equal affinity. The MG ligand consists of three phenyl rings emanating from a central carbon, while TMR has two of the three rings connected by an ether linkage. The binding pockets for MG and TMR in the aptamer, known from high-resolution structures, differ only in the conformation of a few nucleotides. Herein, we applied isothermal titration calorimetry (ITC) to compare the thermodynamics of binding of MG and TMR to the aptamer. Binding heat capacities were obtained from ITC titrations over the temperature range of 15-60 °C. Two temperature regimes were found for MG binding: one from 15 to 45 °C where MG bound with a large negative heat capacity and an apparent stoichiometry (n) of ~0.4 and another from 50 to 60 °C where MG bound with a positive heat capacity and an n of ~1.1. The binding of TMR, on the other hand, revealed only one temperature regime for binding, with a more modest negative heat capacity and an n of ~1.2. The large difference in heat capacity between the two ligands suggests that significantly more conformational rearrangement occurs upon the binding of MG than that of TMR, which is consistent with differences in solvent accessible surface area calculated for available ligand-bound structures. Lastly, we note that the binding stoichiometry of MG was improved not only by an increase in the temperature but also by a decrease in the concentration of Mg(2+) or an increase in the time between ITC injections. These studies suggest that binding of a dynamical ligand to a functional RNA requires the RNA itself to have significant dynamics.

Noncoding RNAs serve myriad functions in the cell, but their biophysical properties are not well understood. Calorimetry offers direct and label-free means for characterizing the ligand-binding and thermostability properties of these RNA. We apply two main types of calorimetry--isothermal titration calorimetry (ITC) and differential scanning calorimetry (DSC)--to the characterization of these functional RNA molecules. ITC can describe ligand binding in terms of stoichiometry, affinity, and heat (enthalpy), while DSC can provide RNA stability in terms of heat capacity, melting temperature, and folding enthalpy. Here, we offer detailed experimental protocols for studying such RNA systems with commercially available conventional and high-throughput ITC and DSC instruments.

Biological RNAs, like their DNA counterparts, contain helical stretches, which have standard Watson-Crick base pairs in the anti conformation. Most functional RNAs also adopt geometries with far greater complexity such as bulges, loops, and multihelical junctions. Occasionally, nucleobases in these regions populate the syn conformation wherein the base resides close to or over the ribose sugar, which leads to a more compact state. The importance of the syn conformation to RNA function is largely unknown. In this study, we analyze 51 RNAs with tertiary structure, including aptamers, riboswitches, ribozymes, and ribosomal RNAs, for number, location, and properties of syn nucleobases. These RNAs represent the set of nonoverlapping, moderate- to high-resolution structures available at present. We find that syn nucleobases are much more common among purines than pyrimidines, and that they favor C2'-endo-like conformations especially among those nucleobases in the intermediate syn conformation. Strikingly, most syn nucleobases participate in tertiary stacking and base-pairing interactions: Inspection of RNA structures revealed that the majority of the syn nucleobases are in regions assigned to function, with many syn nucleobases interacting directly with a ligand or ribozyme active site. These observations suggest that judicious placement of conformationally restricted nucleotides biased into the syn conformation could enhance RNA folding and catalysis. Such changes could also be useful for locking RNAs into functionally competent folds for use in X-ray crystallography and NMR.

Traumatic brain injury features deficits are often ameliorated by dopamine (DA) agonists. We have previously shown deficits in striatal DA neurotransmission using fast scan cyclic voltammetry after controlled cortical impact (CCI) injury that are reversed after daily treatment with the DA uptake inhibitor methylphenidate (MPH). The goal of this study was to determine how a single dose of MPH (5 mg/kg) induces changes in basal DA and metabolite levels and with electrically evoked overflow (EO) DA in the striatum of CCI rats. MPH-induced changes in EO DA after a 2-week daily pre-treatment regime with MPH was also assessed. There were no baseline differences in basal DA or metabolite levels. MPH injection significantly increased basal [DA] output in dialysates for control but not injured rats. Also, MPH injection increased striatal peak EO [DA] to a lesser degree in CCI (176% of baseline) versus control rats (233% of baseline). However, daily pre-treatment with MPH resulted in CCI rats having a comparable increase in EO [DA] after MPH injection when compared with controls. The findings further support the concept that daily MPH therapy restores striatal DA neurotransmission after CCI.

The therapeutic benefits of dopamine (DA) agonists after traumatic brain injury (TBI) imply a role for DA systems in mediating functional deficits post-TBI. We investigated how experimental TBI affects striatal dopamine systems using fast scan cyclic voltammetry (FSCV), western blot, and d-amphetamine-induced rotational behavior. Adult male Sprague-Dawley rats were injured by a controlled cortical impact (CCI) delivered unilaterally to the parietal cortex, or were naïve controls. Amphetamine-induced rotational behavior was assessed 10 days post-CCI. Fourteen days post-CCI, animals were anesthetized and underwent FSCV with bilateral striatal carbon fiber microelectrode placement and stimulating electrode placement in the medial forebrain bundle (MFB). Evoked DA overflow was assessed in the striatum as the MFB was electrically stimulated at 60 Hz for 10 s. In 23% of injured animals, but no naïve animals, rotation was observed with amphetamine administration. Compared with naïves, striatal evoked DA overflow was lower for injured animals in the striatum ipsilateral to injury (p < 0.05). Injured animals exhibited a decrease in V(max) (52% of naïve, p < 0.05) for DA clearance in the hemisphere ipsilateral to injury compared with naïves. Dopamine transporter (DAT) expression was proportionally decreased in the striatum ipsilateral to injury compared with naïve animals (60% of naïve, p < 0.05), despite no injury-related changes in vesicular monoamine transporter or D2 receptor expression (DRD2) in this region. Collectively, these data appear to confirm that the clinical efficacy of dopamine agonists in the treatment of TBI may be related to disruptions in the activity of subcortical dopamine systems.

A liquid-phase radioimmunoassay was used to quantitate sperm-associated IgG in a population of patients with unexplained infertility. Plasma IgG antisperm antibody activity was identified in a subpopulation of these patients. Using the human sperm/hamster egg assay, a unique functional test of human sperm penetrating capacity, we identified a subset of these positive patients whose plasma significantly and reproducibly inhibited penetration. This study implies that antigen-antibody reactions may prevent sperm responses that are critical to sperm-egg fusion.

In an attempt to maximize the success of in vitro fertilization (IVF) and embryo transfer (ET) as a treatment for human infertility, we have examined the relationship of follicular size and number to the rates of oocyte recovery, fertilization, cleavage, and ET in clomiphene citrate-stimulated cycles. The recovery of oocytes from follicles less than 20 mm in diameter was significantly reduced over that from larger follicles, and those oocytes that were obtained from smaller follicles showed a significantly lower rate of fertilization and cleavage. In addition, the overall chance that a patient would undergo ET was greater in a cycle in which more than one follicle 20 mm or larger was developing than in a cycle in which a single large follicle was developing. This latter observation suggests that attempts at laparoscopic oocyte retrieval should be confined to cycles in which more than one accessible large follicle is developing, thereby maximizing the success rate while minimizing the risk and expense for the patient.

Monoclonal antibodies (McAb) against human ejaculated sperm were developed from mice immunized with sperm membrane preparations. A solid-phase radioimmunoassay, with dried sperm as antigen, was employed in McAb screening. The tissue and species specificity of monoclonal antibodies HS 2, 4 and 6 were evaluated after absorption of antibody preparations with heterologous sperm, human serum or seminal plasma or cells from other human organs. The sensitivity of HS 2, 4 and 6 antigens to trypsin exposure was determined: HS 4 antigen was highly sensitive while HS 2 and 6 were not. The regional distribution of McAb 4 on intact sperm cells was determined by immunofluorescence staining. HS 4 may be a sperm-coating antigen based on its presence on sperm and in seminal plasma. This possibility led to an investigation of its role in sperm capacitation. HS 4 antibody binding was reduced when capacitated sperm were compared with noncapacitated cells. HS 4 antibody, when present during capacitation and insemination, was without effect on sperm motility or fusion with zona-free hamster eggs. Trypsin removal of as much as 60% of HS 4 antigen from the cell population also did not impact on sperm function. To identify the molecular correlate of HS 4 antigen, membrane components were extracted from washed sperm with Nonidet P-40, concentrated by acetone precipitation and analyzed electrophoretically in SDS-urea on 10% polyacrylamide slab gels. Immunoassays on protein blots with peroxidase-coupled second antibody identified a single reactive species in the molecular weight range of 130,000. Multiple reactive components were detected in blot transfers of seminal plasma.

We compared fertility potential measurements by the zona-free hamster egg bioassay with the in vitro fertilization of human eggs. Sperm from 24 husbands participating in an in vitro fertilization-embryo transfer program were used in 27 inseminations of the wives' eggs and in simultaneous inseminations of hamster eggs. In seven cases, a positive fertility assessment was obtained in the absence of fertilization of the wives' eggs attributable to egg immaturity or in one case to equipment failure. In the remaining 20 cases, at least one egg was fertilized, for a level of 73%. In 90% of the husbands who fertilized their wives' eggs, a positive fertility assessment was obtained. Two husbands gave false-negative assessments. To account for these, alternative culture conditions were examined; sperm exposure to longer preincubations or at higher concentrations usually improved assessments. These results indicate that fertility assessment with this bioassay is highly correlated with the fertilization of human eggs in vitro.

During a 6-month period, a combination of gamete intrafallopian transfer (GIFT) and in vitro fertilization (IVF) was offered to all couples beginning an IVF treatment cycle in whom the wife had anatomically normal fallopian tubes. It was recommended to these couples that sufficient oocytes be reserved for insemination in vitro to determine whether the husband's spermatozoa could fertilize the wife's oocytes. During this interval, 16 couples underwent the combined IVF-GIFT procedure. All of the IVF-GIFT couples had at least two oocytes inseminated in vitro and at least two oocytes for GIFT. Of the 16 IVF-GIFT couples, only 1 (6.25%) achieved a clinical pregnancy. More important, 50% (8/16) of the IVF-GIFT couples had no oocytes fertilized in vitro. With the information concerning lack of fertilization in vitro, appropriate recommendations concerning future fertility management can be made. If the same couples had undergone the GIFT procedure alone, without additional oocytes fertilized in vitro, this information would not have been obtained.

Human cervical mucus was collected from seven donors during the follicular, ovulatory and luteal phases of the ovulatory menstrual cycle. Individual mucus samples were solubilized and fractionated on Sepharose columns into excluded mucins and low-molecular-weight proteins. Mucin fractions were highly purified, as evidenced by the presence of a single N-terminal amino acid residue, threonine, and by the absence of contaminating plasma proteins. Amino acid compositions of mucins isolated during different menstrual phases of a single donor or from different donors were similar. Mucin carbohydrate compositions were also similar, except for the sialic acid-to-fucose ratio, which varied significantly between donors but not within the menstrual cycle of a single donor. An analysis of variance was applied to evaluate the contribution of mucin composition to viscoelasticity, as quantitated by microrheometry. Viscoelasticity was dependent on the donor, on the percent nondialyzable solids and on the mucin content, b ut not on the phase of the menstrual cycle during which the sample was collected. These findings suggest that mucus function (viscoelasticity) is reflected in carbohydrate composition and/or structure and that this relationship is unique for each donor. Furthermore, the absence of menstrual phase-dependent differences in mucins suggests that mucin concentration and not composition changes in response to alterations in the hormonal milieu.

Human sperm become capable of penetrating zona-free hamster eggs after preincubation in an appropriate culture medium. This observation has led to the development of an assay for characterizing the fertilizing capacity of human spermatozoa. In the present study, the incorporation of sperm by zona-free hamster eggs was quantitated, and several parmeters that contribute to penetration were evaluated. The importance of the motile sperm concentration was established; no penetration was seen at concentrations lower than 6 x 10(5) motile cells/ml, whereas above this level the mean number of incorporated sperm per egg was linearly related to concentration. Freeze-thawed sperm, although capable of penetrating zona-free hamster eggs, did so with lesser frequency than did fresh sperm at equal concentrations of motile sperm. Kinetic experiments indicated that eggs were maximally penetrated after 5 hours of exposure to capacitated sperm and that the cessation in sperm incorporation seen at this time resulted from egg-related changes that occurred during aging in vitro. A protocol for evaluating "fertilizing capacity" of human sperm samples was outlined incorporating the findings from the present study. Using these conditions, reproducible penetration levels were obtained when several ejaculates obtained from the same donor over a 3-month interval were tested at similar motile sperm concentrations.

Human sperm fertility potential, expressed as a quality index (QI), was evaluated objectively from considerations of sperm velocity, percentage of motile forms, sperm density, and ejaculate volume. Turbidimetry was applied in the quantitation of sperm velocity. High QIs (700) were characteristic of semen samples that were capable of penetrating cervical mucus in capillary tube penetration tests, while low QIs (80) were associated with specimens that did not penetrate mucus. The rate of decline in QI as a function of time postejaculation was determined for samples stored at 37 degrees C and at ambient temperature, providing correction factors for the comparative evaluation of semen samples analyzed at different times. The penetrability of cervical mucus was determined by capillary tube penetration testing. Subsequent chemical analysis of sperm-penetrable and impenetrable samples indicated that the concentrations of mucus nondialyzable solids (NDS), mucins, and soluble proteins were significantly higher in impenetrable specimens. These characteristics were also observed in impenetrable mucus obtained from patients with cervical factor infertility. These studies indicate that it is now feasible to employ mucus [NDS] in the diagnosis of cervical factor infertility and turbidimetry in the evaluation of human sperm quality.

The interaction of sperm with the zona-free hamster egg was studied. Hamster sperm were capacitated in Tyrodes media, containing 50% heat-inactivated serum and used to inseminate zona-free eggs in BWW containing 10% serum. Capacitated sperm began fusing with eggs within the first hour of insemination and by 3 h penetration had ceased as indicated by the absence of further changes in the mean number of sperm incorporated per egg. Penetration by capacitated hamster sperm was linearly related to the log of the motile sperm concentration at concentrations above 10(4) cells/ml. The viability of sperm and eggs in culture was limited in these studies to approximately 3-5 h. The existence of a block to unlimited sperm penetration of the zona-free egg was sought in reinsemination experiments. A relatively low sperm concentration was used to initiate egg responses, followed, at timed intervals, by reinsemination with a high challenge concentration of sperm. Subsequent polyspermy levels reflected the presence or absence of the egg's block to polyspermy response. In order to circumvent the problems arising from the rapid aging of hamster sperm in culture, mouse sperm were employed, a convenience afforded by the lack of species specificity in this egg. Reinseminated eggs incorporated additional sperm during the challenge; therefore, the hamster egg is not capable of preventing unlimited sperm penetration. The implications of these findings to the use of the zona-free hamster egg test in fertility evaluation is discussed.

A turbidimetric method has been developed for determining rapidly the fraction of sperm in human ejaculates which show the most vigorous motility. The method is based on the fact that sperm cells so endowed will be the first to swim upward into clear medium from a concentrated cell suspension at the bottom of an optical cuvette. This results in a time-dependent increase in turbidity in the medium which is recorded spectrophotometrically as an increase in absorbance. The determination requires 10 minutes and yields both the fraction of rapidly moving sperm, FRM, and their average velocity, VRM. Examination of 25 samples yielded FRM values of 10% or lower, whereas values for VRM averaged about 100 microns/second. These vigorously motile cells may be the best candidates for fertilization, and samples with a high fraction of such cells should have high fertilizing capacity. It is suggested that this simple turbidimetric test be used in evaluation of human semen as a possible indicator of fertilizing capacity.

A critical aspect of human in vitro fertilization (IVF) treatment programs is the timing of human chorionic gonadotropin (hCG) administration to induce final oocyte maturation. Patients were treated with clomiphene citrate (Serophene, Serono Laboratories, Inc., Randolph, MA), 100 mg, and human menopausal gonadotropin (Pergonal, Serono Laboratories, Inc.) 1 ampule daily from cycle days 4 through 8. The baseline estradiol (E2) level was determined by averaging the E2 values from cycle days 3 and 4. The first day of E2 rise was defined as the day that the E2 level exceeded 150% of the baseline. In general, 5000 IU of hCG (Profasi-HP, Serono Laboratories, Inc.) was administered on the evening of the sixth day of E2 rise. Significantly fewer oocytes were recovered per patient when the hCG was administered on the seventh day of E2 rise, compared with the fifth or sixth day. The recovered oocytes in the seventh-day E2 group had a significantly lower fertilization rate, compared with the other groups. The use of E2-rise days proved to be a simple, successful technique for the timing of hCG administration in an IVF treatment program.