Number Of EPCs Research Articles

Abstract 5524: Progenitor Cell Mobilization is FGF-2 Dependant and Results in the Activation of Osteoclasts

Background Fibroblast growth factor-2 (FGF2) regulates proliferation of bone marrow (BM) stromal cells and has pro-angiogenic effects regulating endothelial cell growth, migration, and reendothelialization. Here we determine the underlying molecular mechanisms of FGF2-dependent statin-mediated progenitor cell mobilization. Methods and Results To determine the molecular mechanisms of FGF2-mediated progenitor cell mobilization, FGF2−/− and wild type (wt) mice were treated with atorvastatin (10mg/kg body weight s.c. per day) and placebo. In contrast to wt mice, the number of Sca1/flk-1 positive EPC in peripheral blood (flow cytometry) of atorvastatin treated FGF2−/− mice did not increase. This was due to an accumulation of Sca1/flk-1 positive cells within the BM. Inhibition of EPC mobilization in FGF2−/− mice was associated with a delay in reendothelialization in a mouse model of a focal endothelial cell denudation and a severe reduction in neoangiogenesis despite statin treatment. BM transplantation experiments demonstrated that reconstitution of FGF2−/− mice with wt stem cells completely restored statin-mediated EPC mobilization while wt mice reconstituted with FGF2−/− stem cells developed a severe mobilization defect associated with an impaired endothelial dependent vasorelaxation (organ chamber) and enhanced neointima formation. In wt mice, statin treatment was associated with an increased number of activated osteoclasts compared to placebo treated mice which are known to play a pivotal role in stem cell mobilization. Treatment with osteoclast-activating RANKL induced an effective mobilization of Sca1/flk1 and Sca1/CD117 positive progenitor cells and enhanced the number of early outgrowth EPC. Conclusion Medullary FGF2 is essential in the statin-mediated mobilization of EPC from BM into peripheral blood. A reduction in the number and activation status of osteoclasts may account for the impairment in EPC mobilization.

Endothelial progenitor cells modulated by IL-1β in multiple organ dysfunction syndrome in porcine

Objective To investigate the modulation of EPCs by interleukin 1β (IL-1β) and p38 mitogen activated protein kinase (p38MAPK) and the pathogenesis resulting from their dysdifferenfiation after trauma.Method Thirty pigs were divided into a control group (n = 15) and a multiple organ dysfimction syndrome (MODS) group (n = 15), the latter of which were subjected to a two-hit injury including hemon'hagic shock and endotoxemia. Phosphorylation of p38MAPK in peripheral blood mononuclear cells was monitored by western blotting. The concentration of IL-1β in peripheral blood plasma was determined by ELISA and the numbers of EPCs with FCM in peripheral blood plasma were monitored. The morbidity rates in the two groups were compared by chi square test. The levels of phosphorylation of p38MAPK in peripheral blood mononuclear cells, the concentmtions of IL-1β in peripheral blood plasma and the numbers of EPCs in the peripheral blood were compared between groups using with Student's t lest. Results The level of p38MAPK phosphorylation was more augmented and the concen-tration of IL-1β higher in peripheral blood mononuelear cells and plasma from MODS pigs compared with those from control pigs; nevertheless the mauler of EPC conspicuously decreased in the peripheral blood (P <0.01). The morbidity rate in the MODS group was much higher than that in the control group (P < 0.01). There were fewer EPCs in the peripheral blood of animals in group M than in the peripheral blood of animals in group C (P <0.01). Conclusions p38MAPK phosphorylation is important for the pathogenesis of MODS. p38MAPK phospho-rylation might cause the concentration of IL-1β in the peripheral blood plasma to rise and could cause a drop in the numbers of EPCs, thereby aggravating the inflanmmatory reaction in MODS. Key words: Multiple organ dysfunction syndrome; p38 mitogen activated protein kinase; Interleukin-1β; En-dothelial progenitor cell; In vivo

SDF1 Gene Variation Is Associated with Circulating SDF1α Level and Endothelial Progenitor Cell Number–The Bruneck Study

BackgroundStromal cell-derived factor-1 (SDF1) and its receptor CXC chemokine receptor 4 (CXCR4) play a critical role in progenitor cell homing, mobilization and differentiation. It would be interesting to assess the predictive value of SDF-1alpha level for EPC number, and to ascertain whether there is a relationship between SDF1 gene variation, plasma SDF-1alpha level, and the number and function of circulating EPCs. We also tested whether EPC number and function was related to CXCR4 gene variation.Methodology and Principal FindingsWe genotyped a cohort of individuals who participated in the Bruneck Study for single nucleotide polymorphisms (SNPs) in the SDF1 and CXCR4 genes, and measured blood SDF1α level as well as EPC number and function. SDF1α levels were correlated with age, gender, alcohol consumption, circulating reticulocyte numbers, and concentrations of matrix metalloproteinase-9, C-reactive protein, cystatin C, fibrinogen and homocytein. In blood samples taken in 2005, EPC number was inversely associated with SDF1α level (p<0.001). EPC number in 2005 was also inversely associated with SDF1α level in 2000 (p = 0.009), suggesting a predictive value of plasma SDF1α level for EPC number. There was an association between the SDF1 gene rs2297630 SNP A/A genotype, increased SDF1α level (p = 0.002) and lower EPC number (p = 0.006).ConclusionsOur data indicate that a SDF1 gene variation (rs2297630) has an influence on SDF1α level and circulating EPC number, and that plasma SDF1α level is a predictor of EPC number.

Review: Endothelial progenitor cells: markers of vascular reparative capacity

Assessment of the propensity for vascular events has been based on measurement of risk factors predisposing one to vascular injury. These assessments are based on the strong associations between risk factors such as hypertension, cholesterol levels, smoking, and diabetes which were first described almost a half century ago. The more recent discovery of the relationship between ongoing inflammation and clinical outcomes has led to a variety of blood-based assays which may impart additional knowledge about an individual's propensity for future cardiovascular events. Vascular health is now better represented as a balance between ongoing injury and resultant vascular repair, mediated at least in part by circulating endothelial progenitor cells. To date, one's risk for vascular events has focused exclusively on assessing propensity for vascular damage, either by assessing conventional risk factors which were initially identified over half a century ago, or more recently by assessing markers of inflammation and other circulating factors which area related to subsequent clinical events. Circulating endothelial progenitor cells play important roles in accelerating endothelialization at areas of vascular damage, and EPC enumeration is a viable strategy for assessing reparative capacity. To date, EPC numbers have been correlated with the numbers of cardiovascular risk factors, extent of coronary disease, and future cardiovascular events. Given that EPC enumeration and functional characterization represent the only assessment of the reparative side of the balance between damage and renovation, this technique may offer independent and different assessment of propensity to cardiovascular injury, greatly improving risk stratification of patients.

Changes in EPC levels and gene expression of homing factors in exercising human skeletal muscle

In the present study the aim was to investigate the circulating level of EPCs defined by expression of VEGFR2 following an acute bout of exercise, as well as plasma levels and skeletal muscle gene expression of factors hypothesized to be involved in homing of EPCs.10 healthy, moderately active males performed 1 h of cycle exercise. Muscle biopsies from the m. vastus lateralis of both legs were obtained before, directly after and 2 h after the exercise bout. Blood samples for flow cytometric measure of EPC numbers and ELISA measurements of plasma protein levels were retrieved before, directly after, 30 minutes after and 2 h after the exercise bout. The circulating level of EPCs increased robustly after the exercise bout. The plasma level of G‐CSF was significantly increased directly after the bout when adjusted for plasma albumin concentration. ICAM‐1 and VEGFR2 mRNA levels were increased 2 h post exercise. The present results indicate that exercise induces recruitment of EPCs to the circulation and prepares the tissue for homing of these cells.

Low-dose growth hormone administration mobilizes endothelial progenitor cells in healthy adults

ObjectiveEndothelial progenitor cells (EPCs) mobilize from the bone marrow secondary to a stimulus and home to sites of injury, where they differentiate into endothelial cells and contribute to the repair of damaged vasculature. We hypothesized that growth hormone (GH) administration would increase the number of circulating EPCs in adults and thereby represent a mechanism to enhance vascular health. DesignA prospective trial of low-dose GH (0.03mg/kg/week for 4 weeks followed by 0.06mg/kg/week for a maximum of four additional weeks) in 10 healthy adults (6 males and 4 females; mean age 37 years, range 26–65). Primary outcomes measured included the number of circulating EPCs as assessed by colony-forming unit (CFU) assay and flow cytometry. Secondary outcomes included plasma measurements of known mediators of EPC mobilization and indices of nitric oxide (NO). Outcomes were measured at baseline and at study completion. ResultsGH administration increased serum IGF-1 (143ng/mL [IQR 121–164] to 222 [IQR 194–244]; P=0.005). The increase in early-outgrowth EPCs (13 CFU per high-power field [IQR 6–24] to 19 [IQR 13–40]; P=0.005) correlated with the peak IGF-1 after adjustment for the baseline number of early-outgrowth EPCs (r=0.719 [95% CI 0.06, 0.93]; P=0.027). The number of late-outgrowth EPCs as well as CD34+, VEGFR2(KDR)+, and AC133+ cells did not significantly change. Other mediators of EPC mobilization were stable while plasma nitrite trended upwards (1.3μmol/L [IQR 0–2.5] to 3.7 [IQR 2.2–8.9]; P=0.052). ConclusionsGH administration selectively augments the early-outgrowth EPC population in healthy individuals. These findings both support GH replacement in the setting of GH deficiency to maintain vascular integrity and have implications for the use of GH in future regenerative cell-based therapies. Furthermore, the decrease in EPCs observed with aging may in part be explained by the declining somatotropic axis, and thereby contribute to cardiovascular senescence.

Endothelial Progenitor Cells, Cardiovascular Risk Factors, Cytokine Levels and Atherosclerosis – Results from a Large Population-Based Study

BackgroundEPC number and functionality are assumed to reflect the endogenous vascular repair capacity with the EPC pool declining in higher ages and being exhausted by unfavorable life-style and risk factors. This intriguing and clinically highly relevant concept, however, has so far been derived from small case-control studies and patient series.Methodology and Principle FindingsIn the population-based Bruneck Study EPC number and EPC-colony forming units (EPC-CFU) were assessed as part of the fourth follow-up evaluation (2005) in 571 and 542 subjects, respectively. EPC number declined with age (p = 0.013), was significantly lower in women (p = 0.006) and higher in subjects on statin, hormone replacement or ACE inhibitor/angiotensin-receptor blockers, and correlated positively with moderate alcohol consumption. Unexpectedly, a positive relation between EPC number and several vascular risk factors emerged. In a step forward multivariate linear regression analysis EPC number was independently related with SDF1α, MMP-9, triglycerides, alcohol consumption, and Hba1c. EPC-CFU in turn was related to SDF1α and diastolic blood pressure. Moreover, EPC number showed a significant positive association with the Framingham risk score (P = 0.001). Finally, there was an inverse association between EPC number and common carotid artery intima-media thickness (p = 0.02) and the carotid artery atherosclerosis score (p = 0.059).ConclusionsOur population-based data confirm the decline of EPC number with advancing age and lend first epidemiological support to a role of SDF-1α and MMP9 in EPC differentiation, mobilization and homing, but are conflict with the view that EPC number is unfavorably affected by cardiovascular risk factors. EPC number increases with the cardiovascular risk estimated by the Framingham risk score (FRS), which in the absence of similar changes for EPC-CFU. Finally, we demonstrate a significant inverse association between EPC number and extent of carotid atherosclerosis even though this association was only of moderate strength and not entirely consistent in other vascular territories.

Explicit experimental evidence for the effectiveness of proximity as mate‐guarding behaviour in reducing extra‐pair fertilization in the Seychelles warbler

Extra-pair copulations (EPCs; copulations outside the pair bond) are widespread in birds and may result in extra-pair fertilizations (EPFs). To increase reproductive success, males should not only seek to gain EPFs, but also prevent their own females from gaining EPFs. Although males could reduce the number of EPCs by their mates, this does not necessarily mean that they reduce the number of EPFs; indeed several studies have found no association between EPCs and EPFs. Male Seychelles warblers (Acrocephalus sechellensis) follow their partner closely during the period when the pair female is most receptive (fertile period). We show that males that guarded their mates more closely were less likely to have extra-pair young in their nest. This study on the Seychelles warbler is the first to provide explicit experimental evidence that mate guarding is effective in reducing EPFs. First, in territories where free-living males were induced to stop mate guarding during the pair female's fertile period, extra-pair parentage was higher than in the control group. Second, in the experimental group, the probability of having an extra-pair nestling in the nest was positively associated with the number of days during the fertile period for which mate guarding was artificially stopped. Thus, male mate guarding was effective in reducing the risk of cuckoldry.

Protecting the Brain With eNOS

See related article, pages 1132–1140 Nitric oxide (NO) plays a major role in both vascular and neural biology in health and disease. This concept is exemplified when one examines the role of NO in the brain and the cerebral circulation. NO is a potent vasodilator, and blood vessels in the brain are normally exposed to NO from 2 major sources—endothelium and neurons. Acting as a messenger molecule, NO mediates the majority of endothelium-dependent responses in the brain.1 The source of this NO is the endothelial isoform of NO synthase (eNOS). This prominent role for NO has been observed in a variety of blood vessels from multiple species including humans.1–3 Through this signaling mechanism, NO influences resting vascular tone and mediates responses to varied stimuli, including endothelium-dependent agonists and increases in blood flow (Figure).1,4,5 In addition, eNOS inhibits vasoconstrictor responses and cerebral vasospasm6–8 and can affect permeability of cerebral endothelium, the blood-brain barrier (Figure).9 eNOS normally inhibits cerebral vascular growth or hypertrophy (Figure),10 a structural change that can have functional consequences by impairing of maximal vasodilator capacity. Impairment of NO-mediated signaling is a key underlying mechanism of vascular dysfunction in diverse forms of disease and aging.1,11–13 Impairment of these eNOS-dependent mechanisms in the presence of cardiovascular risk factors may contribute to …

DECREASED NUMBER OF ENDOTHELIAL PROGENITOR CELLS AFTER INCUBATION WITH WOUND FLUID TAKEN FROM ELDERLY PATIENTS

Neovascularisation plays an important role in wound healing. Most susceptible to impaired wound healing are elderly patients. Therefore, we investigated the influence of wound fluid on the number of differentiated EPC. These cells play a crucial role in neovascularisation, hence impaired proliferation of EPC in elderly patients at the site of interest could be a reason for higher susceptibility to wound healing problems. METHODS: EPC were isolated from peripheral blood mononuclear cells (PBMC) which were isolated from buffy coat by density gradient centrifugation. PBMC were cultivated for 72 h on fibronectin-coated culture plates in endothelial basal medium, supplemented with 20% wound fluid (WF). WF was isolated from patients at 3, 8 and 24 hours after surgical limb treatment. EPC were identified by staining with DiLDL and FITC labeled Ulex europaeus agglutinin-1 and counted using an fluorescence microscope. Wound fluids were isolated from two groups of patients (n = 6 each) with an average age of 35 +/− 6 vs. 66 +/− 11. Results are presented as mean +/− SEM. U-Test was used for statistical evaluation, p<0.05 was considered significant. RESULTS: The number of EPC after incubation with WF taken from young patients was significantly higher: 126 +/− 51(3 h), 128 +/− 51(8 h), 146 +/− 63(24 h) compared to WF taken from elderly patients: 40 +/− 35(3 h), 41 +/− 49(8 h), 43 +/− 45(24 h). DISCUSSION: In this study we show the impaired potency of WF taken from elderly patients for EPC differentiation compared to WF from young individuals. Concentration analysis of proapoptoic cytokines like TNFá and growth factors like VEGF could interpret these results.

The bone marrow-derived endothelial progenitor cell response is impaired in delayed wound healing from ischemia

Vasculogenesis relies on the recruitment of bone marrow-derived endothelial progenitor cells (BMD EPCs) and is stimulated by tissue-level ischemia. We hypothesized that the BMD EPC response is impaired in ischemic wounds and studied the relationship between BMD EPCs and wound healing. We used transgenic Tie-2/LacZ mice, which carry the beta-galactosidase (beta-gal) reporter gene under Tie-2 promoter control. Wild-type mice were lethally irradiated and reconstituted with Tie-2/LacZ bone marrow. Four weeks later, the mice underwent unilateral femoral artery ligation/excision and bilateral wounding of the hindlimbs. Ischemia was confirmed and monitored with laser Doppler imaging. A subset of mice received incisional vs excisional nonischemic bilateral hindlimb wounds, without femoral ligation. Excisional wound closure was measured by using daily digital imaging and software-assisted calculation of surface area. Ischemia resulted in significantly delayed wound healing and differentially affected the number of BMD EPCs recruited to wound granulation tissue and muscle underlying the wounds. At 3 days postwounding, the granulation tissue of the wound base contained significantly fewer numbers of BMD EPCs in ischemic wounds compared with the nonischemic wounds (P < .05). In contrast, significantly more BMD EPCs were present in the muscle underlying the ischemic wounds at this same time point compared with the muscle under the nonischemic wounds (P < .05). In ischemic wounds, eventual wound closure significantly correlated with a delayed rise in BMD EPCs within the wound granulation tissue (Kendall's correlation, -.811, P = .0005) and was significantly associated with a gradual recovery of hindlimb perfusion (P < .0001). By 7 days postwounding, BMD EPCs were incorporated into the neovessels in the granulation tissue. At 14 days and 75 days, BMD EPCs were rarely observed within the wounds. Granulation tissue of excisional ischemic wounds showed significantly less BMD EPCs 3 days postwounding, in association with significantly delayed wound closure. However, the number of BMD EPCs were increased in ischemic hindlimb skeletal muscle, consistent with the notion that ischemia is a powerful signal for vasculogenesis. To our knowledge, this is the first report identifying a deficit in BMD EPCs in the granulation tissue of ischemic skin wounds and reporting the key role for these cells in both ischemic and nonischemic wound healing.

Mesenchymal stem cells: unknown mechanisms of differentiation

Mesenchymal stem cells: unknown mechanisms of differentiation

Vascular endothelial growth factor-C promotes vasculogenesis, angiogenesis, and collagen constriction in three-dimensional collagen gels

Neovascularization, angiogenesis, and collagen constriction are essential for wound healing. We tested whether vascular endothelial growth factor-C (VEGF-C) can promote collagen constriction, capillary sprouting (angiogenesis), and invasion/migration of bone marrow-derived endothelial progenitor cells into collagen (vasculogenesis). We used a recently characterized three-dimensional collagen matrix assay with either monolayers of human dermal microvascular endothelial cells (HMVECs) or bone marrow-derived endothelial progenitor cells (BMD EPCs), obtained from Tie-2 LacZ transgenic mice, overlaid with an acellular layer and then a cellular layer of collagen embedded with fibroblasts, that were nontransduced or transduced with either LacZ adenoviral vector (Ad5) or VEGF-C/Ad5. The ability of VEGF-C to enhance fibroblast-mediated collagen constriction was measured, and gels overlying HMVECs or BMD EPCs were co-cultured, harvested, and assayed for HMVEC migration, sprouting, and capillary-like formation; gels containing BMD EPCs were assayed for EPC invasion/migration into the collagen extracellular matrix. VEGF-C significantly increased collagen constriction and formation of capillary-like structures with true lumina (P < .05) assessed by von Willebrand factor and VEGF receptor-2 immunoassaying. VEGF-C induced a significant increase in HMVEC migration, tubular polarization, and branching sprouts associated with a significant up-regulation of membrane type 1 matrix metalloproteinase (MT1-MMP) ( P < .05). Fibroblasts were necessary to support BMD-EPC invasion/migration from the monolayer into the collagen. Moreover, fibroblasts overexpressing VEGF-C significantly enhanced EPC invasion/migration ( P < .05) into the extracellular matrix by two-fold, and this effect could not be achieved with equivalent levels of exogenous VEGF-C in the absence of fibroblasts. The addition of a soluble VEGF-C competitor protein only partially inhibited these responses, reducing the EPCs by three-fold, but significant numbers of EPCs still invaded/migrated into the extracellular matrix, suggesting that other fibroblast-specific signals also contribute to the vasculogenic response. Fibroblast-specific expression of VEGF-C promotes collagen constriction by fibroblasts and enhances microvascular endothelial cell migration, branching, and capillary sprouting in association with up-regulating MT1-MMP expression. Fibroblasts are necessary for BMD EPC invasion/migration into collagen, and their overexpression of VEGF-C enhances this fibroblast-mediated vasculogenic effect. Collectively, these findings suggest a role for VEGF-C in multiple biologic steps required for wound healing (angiogenesis, vasculogenesis, and collagen constriction). Ischemic wound healing remains an unsolved problem with no previously identified molecular target for therapeutic intervention. This study demonstrates that VEGF-C overexpression by fibroblasts stimulates multiple biologic processes known to impact wound healing, such as collagen constriction, capillary sprouting, and EPC invasion and migration through extracellular matrix. Most ischemic wounds fail to heal and frequently lead to major limb amputation. Available cytokine ointments are ineffective, and revascularization is often not technically feasible. Even when these procedures are accomplished, many ischemic wounds frequently still do not heal because of multifactorial tissue level impairments in the fibroblastic and neovascularization responses at the wound base. Our findings identify an important role for two novel tissue level targets, dermis-derived fibroblasts and VEGF-C, in collagen constriction, angiogenesis, and postnatal vasculogenesis from BMD EPCs. Thus the findings are particularly relevant to the unsolved clinical problem of ischemic wound healing.

Nitric oxide differentially regulates proliferation and mobilization of endothelial progenitor cells but not of hematopoietic stem cells

To investigate the role of nitric oxide in controlling endothelial progenitor (EPC) and hematopoietic stem cell (HSC) mobilization, wild-type mice, L-NAME treated WT and eNOS-/- mice received either PBS or G-CSF for 5 days. Under unstimulated conditions bone marrow of either L-NAME treated WT and eNOS-/- mice, representing acute and chronic NO-deficiency, showed higher CD34(+)Flk-I+ EPC numbers compared to their WT littermates. Furthermore, CD34(+)Flk-I+ progenitors under NO-deficient conditions showed a higher cell turn over since the proliferation and apoptosis activity under in vivo as well as in vitro conditions were enhanced. In line with this finding bone marrow derived EPC differentiation towards endothelial cells was modulated in an NO-dependent manner. Administration of G-CSF resulted in an increase of EPC within the bone marrow of WT animals with a consecutive release of these cells into the peripheral circulation. Under NO-deficient conditions G-CSF failed to increase EPC numbers. In contrast, the HSC population c-kit(+)Lin- was not influenced by nitric oxide. Thus, NO differentially supports the mobilization of the endothelial committed progenitor subpopulation in bone marrow but does not have an effect on HSC in vivo.

Blood donor white blood cell reduction filters as a source of human peripheral blood–derived endothelial progenitor cells

Neovascularization in tumors, wounds, and sites of ischemic injury occurs by both angiogenesis (proliferation from existing vessels) and by vasculogenesis (differentiation into endothelial cells from circulating endothelial progenitor cells [EPCs]). EPCs can be obtained from marrow, from cord blood, or by ex vivo expansion of human peripheral blood (PB). The ease of obtaining human PB EPCs has led many recent studies to utilize PB EPCs. The ability to obtain large numbers of PB EPCs would greatly facilitate characterization to further our understanding of EPC biology and their application in cellular gene therapy. Peripheral blood mononuclear cells (PBMNCs) from whole blood or from the material obtained from white blood cell (WBC) reduction filters were isolated. The cells from both sources were then cultured separately under defined conditions to quantify EPC yield from each source. The yield of EPCs per million PBMNCs plated was approximately 3.5-fold higher from fresh PB. Because greater numbers of PBMNCs were obtained from each filter, however, the mean yield of EPCs from one filter versus fresh blood was 5.4 million versus 0.4 million, respectively (approx. 14-fold increased yield). The use of WBC reduction filters provides a safe, inexpensive, and readily available source for large numbers of PBMNCs from which culture-expanded EPCs can be generated for further study.

The evolutionary psychology of extrapair sex: The role of fluctuating asymmetry

This study explored evolutionary hypotheses concerning extrapair sex (or EPCs: extrapair copulations). Based on recent notions about sexual selection, we predicted that (a) men's number of EPCs would correlate negatively with their fluctuating asymmetry, a measure of the extent to which developmental design is imprecisely expressed, and (b) men's number of times having been an EPC partner of a woman would negatively correlate with their fluctuating asymmetry. In a sample of college heterosexual couples, both hypotheses were supported. In addition, men's physical attractiveness independently predicted how often they had been an EPC partner. Women's anxious attachment style positively covaried with their number of EPC partners, whereas their avoidant attachment style negatively covaried with their number of EPC partners.

Extra-Pair Copulations in the Red-Billed Gull: Females With High-Quality, Attentive Males Resist

AbstractExtra-pair copulation (EPC) and within-pair copulation (WPC) behaviour of red-billed gulls (Larus novaehollandiae scopulinus) was observed in a breeding colony at Kaikoura Peninsula, New Zealand. Twenty-five females and 19 individually colour-marked males were monitored for six hours per day for twenty days from 20 September to 30 October 1973. Extra-pair copulation attempts were common, amounting to 21% of all copulation attempts, but less than 3% were successful during the fertile period of the female. Ten percent of EPCs were on males. Within-pair mountings were eight times more likely to end in cloacal contact than EPCs. Within-pair and EPC attempts occurred throughout the day, but the frequency of WPC attempts increased markedly towards evening, possibly as an adaptation to ensure last-male sperm precedence. Approximately 80% of females, but only 32% of males, were involved in EPC attempts. Mounting within and between pairs increased in frequency about 15 days prior to laying of the first cgg. Within-pair copulation attempts ceased abruptly after the first egg was laid, but EPC attempts on the female continued well into incubation and amounted to 11% of the total EPC attempts. Seventy-five percent of EPC attempts occurred in the fertile period of the recipient female. Approximately 75% of the EPC attempts by males occurred over a nine-day period; six days prior to and two days after its mate had laid the first egg. No EPC attempts were recorded for males more than seven days after its mate commenced laying. The female had control as to whether the copulation attempt would be successful. In WPC attempts the female ended the mount on 72% of the occasions, whereas all EPC attempts were ended by the female. Approximately 91% of within-pair mountings followed some form of courtship feeding display, and in the remaining 9% the male mounted without courtship preliminaries. In contrast, 97% of extra-pair mountings occurred in the absence of courtship displays. On the two instances where preliminaries occurred, mounting followed courtship feeding solicitation by the female towards the strange male. Potentially high quality females which were being well provisioned in courtship feeding by their mates were at greater risk from EPCs because they were able to spend more time at the nest site than less well provisioned females. Females which were well fed during courtship feeding resisted all EPC attempts and retained their partner the next breeding season. Poorly courtship fed females divorced the next season. One such female solicited an EPC four days prior to the laying of her first egg. There was no evidence to suggest that males performing EPCs were at risk from being cuckolded and the male partners of females experiencing high numbers of EPCs did not respond to the risk by having more WPCs or having more genital contacts per hour. It is considered that EPCs were not a major feature of the mating system for the majority of red-billed gulls. The advantages of EPCs were greater for females than for males and the results support the genetic quality hypothesis. Theoretically if males wanted to maximise their fitness they should attempt EPC's on females nesting earlier than themselves, but this only happened on 17% of the EPC attempts. The high number of WPCs, the increase in frequency of copulations in the evening and high courtship feeding rates are measures that help to ensure paternity of the true mate. It is hypothesized that in species like the red-billed gull which have long-term pair-bonds and invest considerable time and energy in courtship feeding and parental care during incubation and chick rearing it would be more advantageous to strengthen the pair-bond than to philander to increase production. In a mating system such as this, philandering would jeopardize the current reproductive investment and future reproduction because those which change partners are less productive than those which retain their partners and for those which divorce the probability of breeding the next season is lower than for those which retain pair-bonds (MILLS, unpub. data). More successful breeding occurs if the pair-bond has been established for more than one year (MILLS, unpub. data). Thus, in this mating system the ''attentive prosper''.

Confidence of Paternity and Parental Effort in Razorbills

-I quantified parental effort in biparentally caring Razorbills (Alca torda) to test whether variation in male effort was related to variation in their mates' receptivity to extrapair copulations. On average, males contributed approximately equally to their mates in chick feedings and overnight nest attendance. There was, however, marked variation in relative male effort, with the proportion of male feeding relative to their mates' feeding ranging from 16 to 72%. No significant portion of this variation was explained by direct and indirect measures of males' confidence of paternity. Support was equivocal for an alternative hypothesis, that variation in male effort is caused by differential male ability. Male care increased significantly with fighting ability and mating arena attendance, but not with five other measures of male vigor. I propose a second alternative hypothesis-that Razorbills and other long-lived, breeding-site-faithful species may reduce their social status over the long term by provisioning poorly, and that lowered status may decrease fitness. Thus, circumstances may exist under which it is beneficial for males to provide care for offspring that may not be their own. Received 22 April 1991, accepted 10 February 1992. ALTHOUGH many species of monogamous birds perform extra-pair copulations (EPCs; McKinney et al. 1984, Birkhead et al. 1987, Westneat et al. 1990), males in most species provide parental care (Wittenberger and Tilson 1980, Oring 1982). The growing evidence that EPCs often succeed in fertilizing eggs (Sherman and Morton 1988, Morton et al. 1990, Westneat 1990) poses a problem in that individuals should be adapted to avoid investing in unrelated offspring (Trivers 1972, Werren et al. 1980). It has been suggested that when males risk losing paternity, they should invest parentally in proportion to their probability of siring their mates' young (Trivers 1972, Maynard Smith 1977, Craig and Jamieson 1985), a prediction I refer to as the confidence-of-paternity hypothesis. I tested this hypothesis with monogamous Razorbills (Alca torda) in which a high percentage of females actively seek EPCs (Wagner 1992a), even though females require parental assistance from the male. Biparental care is necessary in Razorbills because the chick cannot be left unattended, as it requires brooding (Harris and Birkhead 1985), and because in open sites the chick is very vulnerable to predation (Hudson 1982). Evidence that a single parent 1 Present address: Department of Zoological Research, National Zoological Park, Smithsonian Institution, Washington, D. C. 20008, USA. may be unable to provide sufficient food for a chick was suggested in an experiment that showed that two parents had difficulty feeding two young (Lloyd 1977). Confidence of paternity may be estimated directly by quantifying relevant behaviors in which the male participated or witnessed (M01ler 1988), such as the number of EPC attempts involving his mate in his presence. The number of within-pair copulations (WPCs) a male achieves also may affect his confidence of paternity (M0ller 1988), because the outcome of sperm competition (in domestic fowl) is known to be influenced by the relative quantity of sperm of each male stored by females (Martin et al. 1974). Confidence of paternity also may be estimated indirectly by analyzing the receptivity of females to EPCs witnessed by the observer, but not necessarily by the male, such as the number of EPCs the female accepted, or the total number of EPC attempts males made with the female. Female Razorbills were found to vary considerably in the number of EPCs they accepted (0-7; Wagner 1992a), with 50% of females accepting at least one EPC (Wagner 1991a). Females also varied markedly in their receptivity to EPC, accepting EPCs during 0 to 100% of their opportunities (Wagner 1992a). Two measurable forms of parental effort, chick feeding and overnight nest attendance, appear to be costly to Razorbills. The expense of over-