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A failure in self-tolerance leads to autoimmune destruction of pancreatic β-cells and type 1 diabetes (T1D). Low molecular weight dextran sulfate (DS) is a sulfated semi-synthetic polysaccharide with demonstrated cytoprotective and immunomodulatory properties in vitro. However, whether DS can protect pancreatic β-cells, reduce autoimmunity and ameliorate T1D is unknown. Here we report that DS, but not dextran, protects human β-cells against cytokine-mediated cytotoxicity in vitro. DS also protects mitochondrial function and glucose-stimulated insulin secretion and reduces chemokine expression in human islets in a pro-inflammatory environment. Interestingly, daily treatment with DS significantly reduces diabetes incidence in pre-diabetic non-obese diabetic (NOD) mice, and most importantly, reverses diabetes in early-onset diabetic NOD mice. DS decreases β-cell death, enhances islet heparan sulfate (HS)/heparan sulfate proteoglycan (HSPG) expression and preserves β-cell mass and plasma insulin in these mice. DS administration also increases the expression of the inhibitory co-stimulatory molecule programmed death-1 (PD-1) in T-cells, reduces interferon-γ+ CD4+ and CD8+ T-cells and enhances the number of FoxP3+ cells. Collectively, these studies demonstrate that the action of one single molecule, DS, on β-cell protection, extracellular matrix preservation and immunomodulation can reverse diabetes in NOD mice highlighting its therapeutic potential for the treatment of T1D.

Adaptive β-cell replication occurs in response to increased metabolic demand during insulin resistance. The intracellular mediators of this compensatory response are poorly defined and their identification could provide significant targets for β-cell regeneration therapies. Here we show that glucose and insulin in vitro and insulin resistance in vivo activate protein kinase C ζ (PKCζ) in pancreatic islets and β-cells. PKCζ is required for glucose- and glucokinase activator–induced proliferation of rodent and human β-cells in vitro. Furthermore, either kinase-dead PKCζ expression (KD-PKCζ) or disruption of PKCζ in mouse β-cells blocks compensatory β-cell replication when acute hyperglycemia/hyperinsulinemia is induced. Importantly, KD-PKCζ inhibits insulin resistance–mediated mammalian target of rapamycin (mTOR) activation and cyclin-D2 upregulation independent of Akt activation. In summary, PKCζ activation is key for early compensatory β-cell replication in insulin resistance by regulating the downstream signals mTOR and cyclin-D2. This suggests that alterations in PKCζ expression or activity might contribute to inadequate β-cell mass expansion and β-cell failure leading to type 2 diabetes.

Background/Aims: Increased oxidative stress and decreased immune cell apoptosis have been reported to be important factors in the pathogenesis of Crohn’s disease (CD). Our aim was to characterize the genetic expression of molecules implicated in the regulation of oxidative stress and apoptosis in peripheral white mononuclear cells of 18 healthy volunteers (controls) and 20 patients at the onset of CD (active CD [aCD]): 10 who achieved remission (inactive CD [iCD]) and 10 who did not present a complete and deep response to treatment (aCD-T). Methods: mRNA expression was measured by the Agena MassARRAY quantitative gene expression analysis application. The genes analyzed were Fas-receptor (FASR), Fas-ligand (FASL), signal transducer and activator of transcription 1 (STAT1), nuclear factor kappa-light-chain-­enhancer of activated B cells (NFKB1), apoptosis signal-regulating kinase 1 (ASK1), serine/threonine-protein kinase H1 (PSKH1), ATP-binding cassette sub-family B1 (ABCB1) and peptidylprolyl isomerase D (PPID). Results: During a CD flare, we found specific upregulated expression of the genes STAT1 and PSKH1, whereas ABCB1 and FASL were downregulated. In the patients with iCD, FASR and NFKB1 were upregulated. The expression levels of NFKB1, STAT1 and ABCB1 did not show any difference in patients with aCD at the onset of the disease and after treatment (aCD-T). The expression levels of PPID and ASK1 did not show any differences in the patients with aCD, iCD and the controls. We have also reviewed the cellular function and role of these genes in CD. Conclusions: These findings contribute to improving the understanding of the pathogenesis of CD and highlight potential genes involved.

Crohn's disease (CD) is characterized by transmural inflammation that is most frequently located in the region of the terminal ileum. Although the physiopathological mechanisms of the disease are not yet well defined, the unregulated immune response is associated with high production of reactive oxygen species (ROS). These elements are associated with complex systems known as antioxidant defenses, whose function is ROS regulation, thereby preventing the harmful effects of these elements. However, the presence of an imbalance between ROS production and ROS elimination by antioxidants has been widely described and leads to oxidative stress. In this article, we describe the most significant findings on oxidative stress in the intestinal mucosa and peripheral blood.

Hepatocyte growth factor (HGF) is a mitogen required for β-cell replication during pregnancy. To determine whether HGF/c-Met signaling is required for β-cell regeneration, we characterized mice with pancreatic deletion of the HGF receptor, c-Met (PancMet KO mice), in two models of reduced β-cell mass and regeneration: multiple low-dose streptozotocin (MLDS) and partial pancreatectomy (Ppx). We also analyzed whether HGF administration could accelerate β-cell regeneration in wild-type (WT) mice after Ppx. Mouse islets obtained 7 days post-Ppx displayed significantly increased c-Met, suggesting a potential role for HGF/c-Met in β-cell proliferation in situations of reduced β-cell mass. Indeed, adult PancMet KO mice displayed markedly reduced β-cell replication compared with WT mice 7 days post-Ppx. Similarly, β-cell proliferation was decreased in PancMet KO mice in the MLDS mouse model. The decrease in β-cell proliferation post-Ppx correlated with a striking decrease in D-cyclin levels. Importantly, PancMet KO mice showed significantly diminished β-cell mass, decreased glucose tolerance, and impaired insulin secretion compared with WT mice 28 days post-Ppx. Conversely, HGF administration in WT Ppx mice further accelerated β-cell regeneration. These results indicate that HGF/c-Met signaling is critical for β-cell proliferation in situations of diminished β-cell mass and suggest that activation of this pathway can enhance β-cell regeneration. © 2014 by the American Diabetes Association., This work was supported in part by grants from the National Institutes of Health (DK-067351 and DK-077096) and the Juvenile Diabetes Research Foundation (1-2007-3) to A.G.-O. S.E. was the recipient of a postdoctoral fellowship from the National Institutes of Health T32 Research Training grant (T32DK-07052-32). C.D. was the recipient of a research fellowship from the Lawson Wilkins Pediatric Endocrine Society.

This study aims to analyze, in mice, the long-term effects of delayed fatherhood on reproductive fitness and longevity of offspring. Hybrid parental-generation (F0) males, at the age of 12, 70, 100, and 120 wk, were individually housed with a randomly selected 12-wk-old hybrid female. The reproductive fitness of first-generation (F1) females was tested from the age of 25 wk until the end of their reproductive life. In F1 males, the testing period ranged from the age of 52 wk until death. Breeding F1 females from the 120-wk group displayed interbirth intervals longer than females from the 12-, 70-, and 100-wk groups. Furthermore, F2 pups begotten by F1 studs exhibited weaning weights lower than pups from the 12- and 70-wk groups. Offspring from the 120-wk group exhibited shorter survival times associated with lower incidence of tumorigenesis and higher loss of body weight when approaching death when compared to F1 offspring from younger age-groups. The results indicate that advanced paternal age a...

This study aims to analyze, in mice, the long-term effects of delayed fatherhood on postnatal development, spontaneous motor activity, and learning capacity of offspring. Hybrid parental-generation (F 0 ) males, at the age of 12, 70, 100, and 120 wk, were individually housed with a randomly selected 12wk-old hybrid female. The resulting first-generation (F 1 ) offspring were tested for several developmental and behavioral variables. Cumulative percentage of F 1 pups that attained immediate righting in the 120-wk group was lower than that found in the 12-, 70-, and 100-wk groups. Furthermore, the postnatal day of attaining immediate righting was higher in pups from the 120-wk group when compared to pups from the other age-groups. At the age of 20 wk, F 1 offspring from the 120-wk group displayed lower counts of motor activity than offspring from the 12-, 70-, and 100-wk groups. One week later, a higher percentage of offspring from the 100- and 120-wk groups entered the dark compartment during the retention trial of the passive-avoidance test when compared to offspring from the 12wk group. Offspring from the 120-wk group exhibited also lower step-through latency in the retention trial than offspring from the 12-, 70-, and 100-wk groups. These results show that advanced paternal age at conception has long-term effects on preweaning development, spontaneous motor activity, and reduced passive-avoidance learning capacity of mouse offspring. aging, delayed fatherhood, gamete biology, long-term effects, offspring

This study analyzes the long-term effects of delayed motherhood on reproductive fitness and life expectancy of offspring in the mouse. Hybrid (C57BL/6JIco × CBA/JIco) first-generation (F1) females, either at the age of 10 or 51 wk, were individually housed with a randomly selected 12- to 14-wk-old hybrid male following a breeding pen system until females reached the end of their reproductive life. Reproductive fitness of second-generation (F2) females was tested from the age of 25 wk until the end of their reproductive life. In F2 males, the testing period ranged from the age of 52 wk until their natural death. Delayed motherhood of hybrid F1 female mice was associated with a decreased percentage of male F3 offspring at birth and lower life expectancy and body weight during adulthood of F2 offspring. There was, however, no evident negative effect of delayed motherhood on several reproductive fitness variables in either male or female F2 offspring. This included between-parturition interval, litte...

© 2015 Elsevier Inc. Tissue effector cells of the monocyte lineage can differentiate into different cell types with specific cell function depending on their environment. The phenotype, developmental requirements, and functional mechanisms of immune protective macrophages that mediate the induction of transplantation tolerance remain elusive. Here, we demonstrate that costimulatory blockade favored accumulation of DC-SIGN-expressing macrophages that inhibited CD8+ Tcell immunity and promoted CD4+Foxp3+ Treg cell expansion in numbers. Mechanistically, that simultaneous DC-SIGN engagement by fucosylated ligands and TLR4 signaling was required for production of immunoregulatory IL-10 associated with prolonged allograft survival. Deletion of DC-SIGN-expressing macrophages invivo, interfering with their CSF1-dependent development, or preventing the DC-SIGN signaling pathway abrogated tolerance. Together, the results provide new insights into the tolerogenic effects of costimulatory blockade and identify DC-SIGN+ suppressive macrophages as crucial mediators of immunological tolerance with the concomitant therapeutic implications in the clinic., Ministerio de Educación y Ciencia SAF2010-15062, SAF2013-48834-R, and Fundación Mutua Madrileña

Insulin resistance, when combined with decreased β-cell mass and relative insufficient insulin secretion, leads to type 2 diabetes. Mice lacking the IRS2 gene (IRS2(-/-) mice) develop diabetes due to uncompensated insulin resistance and β-cell failure. Hepatocyte growth factor (HGF) activates the phosphatidylinositol 3-kinase/Akt signaling pathway in β-cells without recruitment of IRS1 or IRS2 and increases β-cell proliferation, survival, mass, and function when overexpressed in β-cells of transgenic (TG) mice. We therefore hypothesized that HGF may protect against β-cell failure in IRS2 deficiency. For that purpose, we cross-bred TG mice overexpressing HGF in β-cells with IRS2 knockout (KO) mice. Glucose homeostasis analysis revealed significantly reduced hyperglycemia, compensatory hyperinsulinemia, and improved glucose tolerance in TG/KO mice compared with those in KO mice in the context of similar insulin resistance. HGF overexpression also increased glucose-stimulated insulin secretion in IRS2(-/-) islets. To determine whether this glucose homeostasis improvement correlated with alterations in β-cells, we measured β-cell mass, proliferation, and death in these mice. β-Cell proliferation was increased and death was decreased in TG/KO mice compared with those in KO mice. As a result, β-cell mass was significantly increased in TG/KO mice compared with that in KO mice, reaching levels similar to those in wild-type mice. Analysis of the intracellular targets involved in β-cell failure in IRS2 deficiency showed Pdx-1 up-regulation, Akt/FoxO1 phosphorylation, and p27 down-regulation in TG/KO mouse islets. Taken together, these results indicate that HGF can compensate for IRS2 deficiency and subsequent insulin resistance by normalizing β-cell mass and increasing circulating insulin. HGF may be of value as a therapeutic agent against β-cell failure.

Crohn's disease (CD) is characterized by transmural inflammation that is most frequently located in the region of the terminal ileum. Although the physiopathological mechanisms of the disease are not yet well defined, the unregulated immune response is associated with high production of reactive oxygen species (ROS). These elements are associated with complex systems known as antioxidant defenses, whose function is ROS regulation, thereby preventing the harmful effects of these elements. However, the presence of an imbalance between ROS production and ROS elimination by antioxidants has been widely described and leads to oxidative stress. In this article, we describe the most significant findings on oxidative stress in the intestinal mucosa and peripheral blood.

There is increasing interest in oxidative stress being a potential aetiological factor and/or a triggering factor in Crohn's disease, rather than a concomitant occurrence during the pathogenesis of the disease. Recent research has shown that the immune mononuclear cells of Crohn's disease patients are induced to produce hydrogen peroxide (H2O2). Similarly, the regulation of antioxidant enzymes during disease in these cells has been unravelled, showing that SOD (superoxide dismutase) activity and GPx (glutathione peroxidase) activity is increased during active disease and returns to normal in remission phases. However, catalase remains constantly inhibited which supports the idea that catalase is not a redox-sensitive enzyme, but a regulator of cellular processes. ROS (reactive oxygen species) can be produced under the stimulus of different cytokines such as TNFα (tumour necrosis factor α). It has been shown in different experimental models that they are also able to regulate apoptosis and other cellular processes. The status of oxidative stress elements in Crohn's disease and their possible implications in regulating cellular processes are reviewed in the present paper.

This study aims to analyze, in mice, the long-term effects of delayed fatherhood on reproductive fitness and longevity of offspring. Hybrid parental-generation (F(0)) males, at the age of 12, 70, 100, and 120 wk, were individually housed with a randomly selected 12-wk-old hybrid female. The reproductive fitness of first-generation (F(1)) females was tested from the age of 25 wk until the end of their reproductive life. In F(1) males, the testing period ranged from the age of 52 wk until death. Breeding F(1) females from the 120-wk group displayed interbirth intervals longer than females from the 12-, 70-, and 100-wk groups. Furthermore, F(2) pups begotten by F(1) studs exhibited weaning weights lower than pups from the 12- and 70-wk groups. Offspring from the 120-wk group exhibited shorter survival times associated with lower incidence of tumorigenesis and higher loss of body weight when approaching death when compared to F(1) offspring from younger age-groups. The results indicate that advanced paternal age at conception has negative long-term effects on reproductive fitness and longevity of offspring in the mouse model.

This study aims to analyze, in mice, the long-term effects of delayed fatherhood on postnatal development, spontaneous motor activity, and learning capacity of offspring. Hybrid parental-generation (F(0)) males, at the age of 12, 70, 100, and 120 wk, were individually housed with a randomly selected 12-wk-old hybrid female. The resulting first-generation (F(1)) offspring were tested for several developmental and behavioral variables. Cumulative percentage of F(1) pups that attained immediate righting in the 120-wk group was lower than that found in the 12-, 70-, and 100-wk groups. Furthermore, the postnatal day of attaining immediate righting was higher in pups from the 120-wk group when compared to pups from the other age-groups. At the age of 20 wk, F(1) offspring from the 120-wk group displayed lower counts of motor activity than offspring from the 12-, 70-, and 100-wk groups. One week later, a higher percentage of offspring from the 100- and 120-wk groups entered the dark compartment during the retention trial of the passive-avoidance test when compared to offspring from the 12-wk group. Offspring from the 120-wk group exhibited also lower step-through latency in the retention trial than offspring from the 12-, 70-, and 100-wk groups. These results show that advanced paternal age at conception has long-term effects on preweaning development, spontaneous motor activity, and reduced passive-avoidance learning capacity of mouse offspring.

We analyzed the effect of in vitro aging of mouse oocytes in the presence of dithiothreitol (DTT) on relative levels of glutathione S-transferase (GST) activity and thiols in oocytes, and cell number, DNA fragmentation and cellular allocation to the inner cell mass (ICM) and trophectoderm (TE) lineage at the blastocyst stage. Ovulated oocytes from gonadotropin primed hybrid female mice of 6-8 weeks of age were aged in vitro in the presence of 0, 5, 50, or 500 microM DTT for 6 hr prior to insemination. Relative levels of GST activity and thiols in oocytes were determined by confocal laser scanning microscopy, DNA fragmentation using a single-step TUNEL method, and cell allocation to the ICM and TE lineage by blastocyst staining with propidium iodide and Hoechst 33258. Non-aged oocytes exhibited higher relative levels of GST activity and thiols when compared to oocytes aged in the presence of 0, 5, and 50 microM DTT. Day 5 blastocysts from the 5, 50, and 500 microM DTT groups exhibited higher total number of cells, number of ICM cells, and ICM/TE ratio, but lower percentage of number of nuclei with DNA fragmentation/number of ICM cells than blastocyst from the 0 microM DTT group. These data show that DTT counteracts the negative effects of a post-ovulatory aging of mouse oocytes in vitro on relative levels of GST activity and thiols in oocytes, and percentage of number of nuclei with DNA fragmentation/number of ICM cells, total number of cells, number of ICM cells and ICM/TE ratio in Day 5 blastocysts.

Results: Chronic DSS colitis induced persistent thickening of the muscularis propria (MP) with infiltration of vimentin+ cells in the chronic phase but not in acute colitis. Moreover, a mucosal healing response was associated with more cycles and recovery. Myofibroblasts (vimentin+ a-SMA+) were present in the submucosa (SM) in the chronic phase and not in the acute model. Differential deposition of collagen I and III was observed: collagen I was present in the SM around vimentin+ cells. Collagen III deposition in the SM started in the acute phase, further increased in chronic colitis with infiltration of collagen III between smooth muscle cells of the MP, to intestinal stricture formation in CD. During prolonged recovery there was a decrease in collagen III. Tenascin expression was scant in control colon but was clearly present in the mucosa and SM in the acute phase in individual cells. A linear deposition of tenascin around the crypts was also seen as expected in acute colitis. The linear deposition in the mucosa and submucosal spots persisted after 2 cycles of DSS, correlating with persisting tissue damage. The expression of tenascin decreased after 3 cycles and more so after additional recovery, in association with the disappearance of tissue damage. Conclusions: The connective tissue changes observed in chronic murine colitis induced by repeated cycles of DSS reflects tissue remodeling as seen in CD. The technical ease and reproducibility of the DSS model and ability to induce it in mice of a common inbred strain makes it an attractive model to study the connective tissue changes occurring in IBD.

Background: An increase of oxidative stress (OxS) and a decrease of apoptosis have been reported as key factors in Crohn's disease (CD) pathogenesis. We have previously shown that OxS in CD depends on an increased production of H2O2 which detoxification is independent of catalase (CAT) enzyme because CAT showed permanently inhibited in CD patients1. CAT could be more implicated in regulating cellular processes, as apoptosis, than in detoxifying H2O2. Aims: To characterize the genetic expression of CAT and other OxS and apoptosis related genes in CD at beginning of the disease and when remission has been achieved. Methods: Blood samples were obtained from 18 healthy subjects (H), 20 patients at onset of CD and yet to begin any specific medication (active CD, aCD) and from 10 patients who achieved clinical, analytical and morphological remission (inactive CD, iCD). Patients were diagnosed according to Leonard-Jones criteria and disease activity was scored based on the Harvey-Bradshaw index, serological markers of inflammation (CRP, fibrinogen) and evaluation of morphological lesions (magnetic resonance enterography, colonoscopy or capsule endoscopy). PWMC were isolated by Histopaque sedimentation. Total RNA from leukocytes was extracted according to manufacturer's protocol (LeukoLOCKTM; Ambion). mRNA expression was measured by Sequenom's MassARRAY® quantitative gene expression analysis application. The genes analyzed were: CAT, SOD2, NOS2A, STAT1, NFKB1, PKCgamma, PKCzeta, PSKH1, PPID, ABCB1, ASK1, FASR, FASLG, TERT, IL-2, IL23R. Results: Genes differentially expressed in CD are summarized in the table. Specific upregulated genes during flair were SOD2, STAT1, and PSKH1, and down-regulated were FASL and ABCB1. In iCD, FASR and NFKB1 were up-regulated. The down-regulation of CAT gene expression was persistent. Conclusions: Up regulation of SOD2, STAT1 and PSKH1 only during a flare, indicates the relation between OxS and aCD. Down regulation of FASL and ABCB1 shows an apoptotic and multidrug resistance alteration at onset of the disease. Increased expression of FASR and NFKB1 in iCD could indicate that apoptosis are genetically affected in CD. Permanent inhibition of CAT activity in CD patients correlates to a persistent down regulation of the CAT-gene mRNA. The implications of CAT inhibition in regulating cellular processes such as apoptosis should be studied. 1) Beltran B, et al. Inflamm Bowel Dis. 2010 16:76-86.

ileocolonoscopy at diagnosis. Of the 276 patients not receiving an ileocolonoscopy at diagnosis, 80 did in follow-up. CD pts underwent significantly more ileocolonscopies at diagnosis, compared to UC pts (44% vs 17%, p < 0.001) and IBDU pts (44% vs 4.5%, p = 0.001). Reasons for follow-up endoscopy were: relapse of symptoms (76%), evaluating treatment effect and/or disease extension (22%) and cancer surveillance (2.4%). In 71% of the follow-up endoscopies active inflammation was seen, however this led to treatment intensification in only 65%. Conclusions: Endoscopies is an often used tool in IBD patients, although only 29% patients received an ileocolonoscopy at diagnosis. Fourty-five percent of the endoscopies performed at follow-up led to treatment intensification, but in over one third of endoscopies showing inflammation, no adjustments were made.

En la EC existe una resistencia a la apoptosis en lamina propia intestinal, no existen estudios homogeneos en celulas mononucleres en sangre periferica. Objetivo Estudiar la susceptibilidad a la apoptosis de los linfocitos no activados de sangre periferica de pacientes EC, despues de activar la celulas in vitro y anadirles un estimulo apoptotico externo (anticuerpo (Ac) agonista Fas). Metodos Se obtuvo sangre heparinizada de sujetos control (n=11) y de EC al debut y sin medicacion (n=8). Los pacientes fueron diagnosticados siguiendo los criterios Leonard-Jones y la actividad segun el Harvey-Bradshaw. Los linfocitos fueron aislados mediante centrifugacion en gradiente con Histopaque ® , seguido de una purificacion en negativo de los linfocitos T no activados (Depletion Dynabeads ® ). Las celulas asi aisladas, fueron cultivadas durante 5 dias en presencia (o ausencia) de Ac CD3 unido a la placa y Ac CD28 diluido en medio X Vivo-15 (Lonza). Transcurrido este tiempo necesario para activar las celulas, se anadio Ac Fas (1 μg/mL) y transcurridas las incubaciones de 5 y 24 h, se detecto la apoptosis mediante citometria de flujo (Annexin-V). Resultados La activacion de los linfocitos T mediada por la exposicion a los Ac CD3/CD28 resulto en un incremento de la apoptosis, tanto en los controles (52,37±5,2) como en la EC (36,54±3,9) p=0,052. Al anadir el Ac Fas, se incremento el porcentaje de celulas apoptoticas (controles 66,09±3,2 vs EC 53,37±6,2); pero fue necesaria una exposicion de 24 h para que este efecto fuera significativo: controles 69,05±3,7 vs EC 53,35±5,6 (p=0,033). Conclusiones La resistencia a la apoptosis, puede ser una caracteristica que presenten los linfocitos T de sangre periferica antes de alcanzar la mucosa intestinal. Esta alteracion intrinseca en la capacidad apoptotica ayuda a clarificar la relacion entre, respuesta inmune innata y adquirida en la EC y la fisiopatologia de la enfermedad.

The aim of the present study was to determine, in the mouse, whether maintaining females as virgins until an advanced reproductive age was associated with decreased reproductive performance and reproductive lifespan compared with females of the same age that were first mated with males at an earlier reproductive age. Randomly selected virgin hybrid (C57BL/6JIco female × CBA/JIco male) female mice were housed individually with a randomly selected 12- to 14-week-old hybrid male either at the age of 28 weeks (normal breeding group; n = 20) or 51 weeks (delayed breeding group; n = 23) for the rest of their reproductive life. Females were checked once daily to determine the day of parturition and to record the litter size and gender of pups at birth for each consecutive litter. At weaning, offspring were weighed and killed. Delayed breeding was associated with smaller litter sizes, both at birth and at weaning, a higher bodyweight of pups at weaning, a higher percentage of litters with at least one newborn pup cannibalised, earlier cessation of female reproductive life and a higher mortality rate of dams during the breeding period. These results show that delayed breeding in the mouse is associated with decreased reproductive performance and a shorter reproductive lifespan compared with females bred at an earlier reproductive age.