Global genome splicing analysis reveals an increased number of alternatively spliced genes with aging

Alternative splicing (AS) is a key regulatory mechanism for the development of different tissues; however, not much is known about changes to alternative splicing during aging. Splicing events may become more frequent and widespread genome‐wide as tissues age and the splicing machinery stringency decreases. Using skin, skeletal muscle, bone, thymus, and white adipose tissue from wild‐type C57BL6/J male mice (4 and 18 months old), we examined the effect of age on splicing by AS analysis of the differential exon usage of the genome. The results identified a considerable number of AS genes in skeletal muscle, thymus, bone, and white adipose tissue between the different age groups (ranging from 27 to 246 AS genes corresponding to 0.3–3.2% of the total number of genes analyzed). For skin, skeletal muscle, and bone, we included a later age group (28 months old) that showed that the number of alternatively spliced genes increased with age in all three tissues (P < 0.01). Analysis of alternatively spliced genes across all tissues by gene ontology and pathway analysis identified 158 genes involved in RNA processing. Additional analysis of AS in a mouse model for the premature aging disease Hutchinson–Gilford progeria syndrome was performed. The results show that expression of the mutant protein, progerin, is associated with an impaired developmental splicing. As progerin accumulates, the number of genes with AS increases compared to in wild‐type skin. Our results indicate the existence of a mechanism for increased AS during aging in several tissues, emphasizing that AS has a more important role in the aging process than previously known.     

<Emphasis Type="Italic">Lepidium meyenii</Emphasis> (Maca) increases litter size in normal adult female mice

Background  

Lepidium meyenii, known as Maca, grows exclusively in the Peruvian Andes over 4000 m altitude. It has been used traditionally to increase fertility. Previous scientific studies have demonstrated that Maca increases spermatogenesis and epididymal sperm count. The present study was aimed to investigate the effects of Maca on several fertility parameters of female mice at reproductive age.     

Immunoexpression of the relaxin receptor LGR7 in breast and uterine tissues of humans and primates

Background  

The receptor for the peptide hormone relaxin has recently been identified as the heptahelical G-protein coupled receptor, LGR7. In order to generate molecular tools with which to characterize both in vivo and in vitro expression of this receptor in human and primate tissues, specific monotypic antibodies have been generated and applied to a preliminary analysis of human and primate female reproductive tissues.     

Aging impacts isolated lymphoid follicle fevelopment and function

Background  

Immunosenescence is the age-related decline and dysfunction of protective immunity leading to a marked increase in the risk of infections, autoimmune disease, and cancer. The majority of studies have focused on immunosenescence in the systemic immune system; information concerning the effect of aging on intestinal immunity is limited. Isolated lymphoid follicles (ILFs) are newly appreciated dynamic intestinal lymphoid structures that arise from nascent lymphoid tissues, or cryptopatches (CP), in response to local inflammatory stimuli. ILFs promote "homeostatic" responses including the production of antigen-specific IgA, thus playing a key role in mucosal immune protection. ILF dysfunction with aging could contribute to immunosenescence of the mucosal system, and accordingly we examined phenotypic and functional aspects of ILFs from young (2 month old) and aged (2 year old) mice.     

Cumulus expansion is impaired with advanced reproductive age due to loss of matrix integrity and reduced hyaluronan

Reproductive aging is associated with ovulatory defects. Age-related ovarian fibrosis partially contributes to this phenotype as short-term treatment with anti-fibrotic compounds improves ovulation in reproductively old mice. However, age-dependent changes that are intrinsic to the follicle may also be relevant. In this study, we used a mouse model to demonstrate that reproductive aging is associated with impaired cumulus expansion which is accompanied by altered morphokinetic behavior of cumulus cells as assessed by time-lapse microscopy. The extracellular matrix integrity of expanded cumulus–oocyte complexes is compromised with advanced age as evidenced by increased penetration of fluorescent nanoparticles in a particle exclusion assay and larger open spaces on scanning electron microscopy. Reduced hyaluronan (HA) levels, decreased expression of genes encoding HA-associated proteins (e.g., Ptx3 and Tnfaip6), and increased expression of inflammatory genes and matrix metalloproteinases underlie this loss of matrix integrity. Importantly, HA levels are decreased with age in follicular fluid of women, indicative of conserved reproductive aging mechanisms. These findings provide novel mechanistic insights into how defects in cumulus expansion contribute to age-related infertility and may serve as a target to extend reproductive longevity.     

Serial analysis of gene expression reveals differential expression between endometriosis and normal endometrium. Possible roles for AXL and SHC1 in the pathogenesis of endometriosis

Background  

Endometriosis is a clinical condition that affects up to 10% of the women of reproductive age. Endometriosis is characterized by the presence of endometrial tissues outside the uterine cavity and can lead to chronic pelvic pain, infertility and, in some cases, to ovarian cancer.     

Pioglitazone administration alters ovarian gene expression in aging obese lethal yellow mice

Background  

Women with polycystic ovary syndrome (PCOS) are often treated with insulin-sensitizing agents, e.g. thiazolidinediones (TZD), which have been shown to reduce androgen levels and improved ovulatory function. Acting via peroxisome proliferator-activated receptor (PPAR) gamma, TZD alter the expression of a large variety of genes. Lethal yellow (LY; C57BL/6J Ay/a) mice, possessing a mutation (Ay) in the agouti gene locus, exhibit progressive obesity, reproductive dysfunction, and altered metabolic regulation similar to women with PCOS. The current study was designed to test the hypothesis that prolonged treatment of aging LY mice with the TZD, pioglitazone, alters the ovarian expression of genes that may impact reproduction.     

Exendin-4 improves blood glucose control in both young and aging normal non-diabetic mice, possible contribution of beta cell independent effects

Aims

Type 2 diabetes is highly prevalent in the elderly population. Glucagon like Peptide-1 mimetic such as exendin-4 augments post-prandial insulin secretion. However, the potential influence of aging on the therapeutic effects of this peptide has not been well studied. In this study, we examined the glucose regulatory effects of exendin-4 in mice with different ages.

Methods

We treated 3-month and 20 to 22-month old C57/DBA mice with 10 nM/kg exendin-4 for 10 days with measurements of blood glucose and body weight. We performed OGTT and ITT to evaluate the glucose response and insulin sensitivity. Islet morphology and beta cell mass were measured by immuno-staining and beta cell proliferation was evaluated by BrdU incorporation and PCNA staining. Real-time PCR and western blot were used to measure protein changes in the liver tissue after exendin-4 treatment.

Results

Exendin-4 treatment improved glycemic control in both 3-month and 20 to 22-month old mice. In both groups of mice, the blood glucose lowering effect was independent of beta cell function as indicated by unchanged beta cell proliferation, insulin secretion or beta cell mass. Moreover, we found that exendin-4 treatment increased hepatic AKT and FOXO1 phosphorylation and inhibited glucose-6-phosphotase (G6P) and Phosphoenolpyruvate carboxykinase (PEPCK) expression in young mice, but this effect was attenuated in aging mice while the insulin sensitivity showed no change in the young group but significantly improved in aging mice.

Conclusion

Based on these data, we conclude that the glucose lowering effect of exendin-4 in normal non-diabetic mice was not blunted by aging. We further showed that although there was slight difference in the glucose modulating mechanism of exendin-4 therapy in young and aged mice, the improved glucose control seemed uncorrelated with increased beta cell mass or insulin secretion.     

Maternal vitamin d deficiency delays glomerular maturity in f1 and f2 offspring

Background

There is a high prevalence of vitamin D insufficiency in women of reproductive age.

Methods

This work studied the first two generations of offspring (F1 and F2) of Swiss mice from mothers fed one of two diets: SC (standard chow) or VitD- (vitamin D-deficient). Functional and developmental kidney measurements were taken.

Results

The first two generations of the VitD- group had higher blood pressure at 6 months of age than the offspring of the SC group as well as an increase in renin and AT1r expression. However, at all ages, both F1 and F2 VitD- mice had shorter glomerular diameters, and diet played a significant role in the total variation. Both the F1 and F2 generations of the VitD- group had more immature glomeruli than offspring from the SC group. Immature glomeruli begin to disappear at 10 days, but at this age, F1-VitD- mice had more immature and mature glomeruli than F1-SC mice. At 6 months of age, F1-VitD- mice exhibited more glomeruli, while F2-VitD- mice exhibited the same number of glomeruli as F2-SC mice, but fewer glomeruli compared to the F1-VitD group. Both diet and generation account for the total variation in the number of glomeruli. Decreases in urine output and podocin expression and increases in urea and creatinine in the urine were observed in F1 offspring.

Conclusion

These findings demonstrate that maternal vitamin D deficiency accompanies changes in the renal expression of important factors that may retard the maturation of glomeruli by extending the period of nephrogenesis.     

SIRT2 induces the checkpoint kinase BubR1 to increase lifespan

Mice overexpressing the mitotic checkpoint kinase gene BubR1 live longer, whereas mice hypomorphic for BubR1 (BubR1^H/H) live shorter and show signs of accelerated aging. As wild‐type mice age, BubR1 levels decline in many tissues, a process that is proposed to underlie normal aging and age‐related diseases. Understanding why BubR1 declines with age and how to slow this process is therefore of considerable interest. The sirtuins (SIRT1‐7) are a family of NAD⁺‐dependent deacetylases that can delay age‐related diseases. Here, we show that the loss of BubR1 levels with age is due to a decline in NAD⁺ and the ability of SIRT2 to maintain lysine‐668 of BubR1 in a deacetylated state, which is counteracted by the acetyltransferase CBP. Overexpression of SIRT2 or treatment of mice with the NAD⁺ precursor nicotinamide mononucleotide (NMN) increases BubR1 abundance in vivo. Overexpression of SIRT2 in BubR1^H/H animals increases median lifespan, with a greater effect in male mice. Together, these data indicate that further exploration of the potential of SIRT2 and NAD⁺ to delay diseases of aging in mammals is warranted.     

Nontuberculous mycobacterium M. avium infection predisposes aged mice to cardiac abnormalities and inflammation

Biological aging dynamically alters normal immune and cardiac function, favoring the production of pro‐inflammatory cytokines (IL‐1β, IL‐6, and TNF‐α) and increased instances of cardiac distress. Cardiac failure is the primary reason for hospitalization of the elderly (65+ years). The elderly are also increasingly susceptible to developing chronic bacterial infections due to aging associated immune abnormalities. Since bacterial infections compound the rates of cardiac failure in the elderly, and this phenomenon is not entirely understood, the interplay between the immune system and cardiovascular function in the elderly is of great interest. Using Mycobacterium avium, an opportunistic pathogen, we investigated the effect of mycobacteria on cardiac function in aged mice. Young (2–3 months) and old (18–20 months) C57BL/6 mice were intranasally infected with M. avium strain 104, and we compared the bacterial burden, immune status, cardiac electrical activity, pathology, and function of infected mice against uninfected age‐matched controls. Herein, we show that biological aging may predispose old mice infected with M. avium to mycobacterial dissemination into the heart tissue and this leads to cardiac dysfunction. M. avium infected old mice had significant dysrhythmia, cardiac hypertrophy, increased recruitment of CD45⁺ leukocytes, cardiac fibrosis, and increased expression of inflammatory genes in isolated heart tissue. This is the first study to report the effect of mycobacteria on cardiac function in an aged model. Our findings are critical to understanding how nontuberculous mycobacterium (NTM) and other mycobacterial infections contribute to cardiac dysfunction in the elderly population.     

Decline in mitochondrial bioenergetics and shift to ketogenic profile in brain during reproductive senescence

Background

We have previously demonstrated that mitochondrial bioenergetic deficits precede Alzheimer's pathology in the female triple transgenic Alzheimer's (3xTgAD) mouse model. Herein, we sought to determine the impact of reproductive senescence on mitochondrial function in the normal non-transgenic (nonTg) and 3xTgAD female mouse model of AD.

Methods

Both nonTg and 3xTgAD female mice at 3, 6, 9, and 12 months of age were sacrificed and mitochondrial bioenergetic profile as well as oxidative stress markers were analyzed.

Results

In both nonTg and 3xTgAD mice, reproductive senescence paralleled a significant decline in PDH, and Complex IV cytochrome c oxidase activity and mitochondrial respiration. During the reproductive senescence transition, both nonTg and 3xTgAD mice exhibited greater individual variability in bioenergetic parameters suggestive of divergent bioenergetic phenotypes. Following transition through reproductive senescence, enzymes required for long-chain fatty acid (HADHA) and ketone body (SCOT) metabolism were significantly increased and variability in cytochrome c oxidase (Complex IV) collapsed to cluster at a ∼ 40% decline in both the nonTg and 3xTgAD brain which was indicative of alternative fuel generation with concomitant decline in ATP generation.

Conclusions

These data indicate that reproductive senescence in the normal nonTg female brain parallels the shift to ketogenic/fatty acid substrate phenotype with concomitant decline in mitochondrial function and exacerbation of bioenergetic deficits in the 3xTgAD brain.

General significance

These findings provide a plausible mechanism for increased life-time risk of AD in postmenopausal women and suggest an optimal window of opportunity to prevent or delay decline in bioenergetics during reproductive senescence.     

Mycoplasma pneumoniae and/or Chlamydophila pneumoniae inoculation causing different aggravations in cholesterol-induced atherosclerosis in apoE KO male mice

Background  

Chamydophila pneumoniae (CP) and/or Mycoplasma pneumoniae (MP) are two bacteria detected in vulnerable atheromas. In this study we aimed to analyze whether CP and/or MP aggravates atherosclerosis induced by cholesterol-enriched diet in C57BL/6 apoE KO male mice. Thirty male apoE KO mice aged eight weeks fed by a diet containing 1% cholesterol until 32 weeks of age were divided into four groups: the first was inoculated with CP (n = 7), the second with MP (n = 12), the third with both CP + MP (n = 5), and the fourth with saline (sham n = 6). The animals were re-inoculated at 36 weeks of age, and sacrificed at 40 weeks of age. Two ascending aorta and one aortic arch segments were sampled. In the most severely obstructed segment, vessel diameter, plaque height, percentage of luminal obstruction and the degree of adventitial inflammation were analyzed. The plaque area/intimal surface ratio was obtained by measuring all three segments. The adventitial inflammation was semiquantified (0 absent, 1 mild, 2 moderate, and 3 diffuse).     

Selection of reference genes for quantitative real-time PCR expression studies in the apomictic and sexual grass Brachiaria brizantha

Background  

Brachiaria brizantha is an important forage grass. The occurrence of both apomictic and sexual reproduction within Brachiaria makes it an interesting system for understanding the molecular pathways involved in both modes of reproduction. Quantitative real time PCR (qRT-PCR) has emerged as an important technique to compare expression profile of target genes and, in order to obtain reliable results, it is important to have suitable reference genes. In this work, we evaluated eight potential reference genes for B. brizantha qRT-PCR experiments, isolated from cDNA ovary libraries. Vegetative and reproductive tissues of apomictic and sexual B. brizantha were tested to validate the reference genes, including the female gametophyte, where differences in the expression profile between sexual and apomictic plants must occur.     

Mechanical strain modulates age-related changes in the proliferation and differentiation of mouse adipose-derived stromal cells

Background  

Previous studies on the effects of aging in human and mouse mesenchymal stem cells suggest that a decline in the number and differentiation potential of stem cells may contribute to aging and aging-related diseases. In this report, we used stromal cells isolated from adipose tissue (ADSCs) of young (8-10 weeks), adult (5 months), and old (21 months) mice to test the hypothesis that mechanical loading modifies aging-related changes in the self-renewal and osteogenic and adipogenic differentiation potential of these cells.     

Redundancy of myostatin and growth/differentiation factor 11 function

Background  

Myostatin (Mstn) and growth/differentiation factor 11 (Gdf11) are highly related transforming growth factor β (TGFβ) family members that play important roles in regulating embryonic development and adult tissue homeostasis. Despite their high degree of sequence identity, targeted mutations in these genes result in non-overlapping phenotypes affecting distinct biological processes. Loss of Mstn in mice causes a doubling of skeletal muscle mass while loss of Gdf11 in mice causes dramatic anterior homeotic transformations of the axial skeleton, kidney agenesis, and an increase in progenitor cell number in several tissues. In order to investigate the possible functional redundancy of myostatin and Gdf11, we analyzed the effect of eliminating the functions of both of these signaling molecules.     

Expression of costimulatory molecules in the bovine corpus luteum

Background  

A major event in the post-meiotic development of male germ cells is the formation of the acrosome. This process can be perturbed in C57BL/6 mice by administration of the small molecule miglustat (N-butyldeoxynojirimycin, NB-DNJ). The miglustat-treated mice produce morphologically abnormal spermatozoa that lack acrosomes and are poorly motile. In C57BL/6 mice, miglustat can be used to maintain long-term reversible infertility. In contrast, when miglustat was evaluated in normal men, it did not affect spermatogenesis. To gain more insight into this species difference we have now evaluated the reproductive effects of miglustat in rabbits, in multiple mouse strains and in interstrain hybrid mice.     

Oocyte production and sperm utilization patterns in semi-fertile strains of <Emphasis Type="Italic">Caenorhabditis elegans</Emphasis>

Background  

Caenorhabditis elegans hermaphrodites are capable of producing hundreds of progeny. However, genetic and environmental factors can keep many animals from attaining their full reproductive potential. In these situations, efficient use of any functional gametes becomes more important for reproductive success. To learn about this aspect of C. elegans reproductive biology, we examined oocyte production and sperm utilization patterns in a unique collection of semi-fertile sperm function mutants.