Aging in the Syrian hamster testis: Inflammatory-oxidative status and the impact of photoperiod.

Testicular aging is linked to histological, morphological and functional alterations. In the present study, we investigated whether aging affects the inflammatory and oxidative status in the testis by comparing young adult, middle-aged adult and aged hamsters. The Syrian hamster, a thoroughly studied seasonal breeder, was chosen as the experimental model since it allows further investigations on the role of photoperiod and melatonin in testicular aging with a minimal impact of the experimental intervention on the animal well-being and the subsequent results achieved. In testes of aged hamsters, we found a decrease in melatonin concentration, a thickening of the wall of the seminiferous tubules as well as a significant increase in IL-1ß, NLRP3 and cyclooxygenase 2 expression, PGD2 production, macrophages numbers, lipid peroxidation and anti-oxidant enzyme catalase levels. Interestingly, when aged hamsters were transferred from a long day (LD) to a short day (SD) photoperiod for 16 weeks, testicular melatonin concentration increased while local inflammatory processes and oxidative stress were clearly reduced. Overall, these results indicate that melatonin might display anti-inflammatory and anti-oxidant capacities in the aged testes.

Ageing and inflammation in the male reproductive tract.

Ageing is usually characterised by a mild chronic proinflammatory state. Despite the tight association between both processes, the phenomenon has recently been termed inflammageing. Inflammation in the male reproductive tract is frequently linked with bacterial or virus infections but also with a broad range of noninfectious processes. Prostatitis, epididymitis and orchitis, among others, can lead to infertility. However, in spite of the inflammation theory of disease, chronic inflammation in male urogenital system does not always cause symptoms. With advancing age, inflammatory processes are commonly observed in the male reproductive tract. Nevertheless, the incidence of inflammation in reproductive organs and ducts varies greatly among elderly men. Inflammageing is considered a predictor of pathogenesis and the development of age-related diseases. This article briefly summarises the current state of knowledge on inflammageing in the male reproductive tract. Yet, the precise aetiology of inflammageing in the male urogenital system, and its potential contribution not only to infertility but most importantly to adverse health outcomes remains almost unknown. Thus, further investigations are required to elucidate the precise cross-links between inflammation and male reproductive senescence, and to establish the impact of anti-inflammatory drug treatments on elder men's general health status.

Ovarian aging and the activation of the primordial follicle reserve in the long-lived Ames dwarf and the short-lived bGH transgenic mice.

The aim of this study was to evaluate the effect of growth hormone (GH) in the maintenance of the ovarian primordial follicle reserve. Ovaries from 16 mo old GH-deficient Ames Dwarf (df/df) and Normal (N/df) mice were used. A subgroup of df/df and N mice received GH or saline injections for six weeks starting at 14 mo of age. In addition, ovaries from 12 mo old mice overexpressing bovine GH (bGH) and controls were used. df/df mice had higher number of primordial and total follicles than N/df mice (p < 0.05), while GH treatment decreased follicle counts in both genotypes (p < 0.05). In addition, bGH mice had lower number of primordial and total follicles than the controls (p < 0.05). pFoxO3a levels were higher in mice treated with GH and in bGH mice (p < 0.05) when comparing with age match controls. These results indicate that increased circulating GH is associated with a reduced ovarian primordial follicle reserve and increased pFoxO3a content in oocytes.

Growth hormone actions during development influence adult phenotype and longevity.

There is considerable evidence that exposure to undernutrition, overnutrition, stress or endocrine disruptors during fetal development can increase the probability of obesity, hypertension, cardiovascular disease and other problems in adult life. In contrast to these findings, reducing early postnatal growth by altering maternal diet or number of pups in a litter can increase longevity. In hypopituitary Ames dwarf mice, which are remarkably long lived, a brief period of growth hormone therapy starting at 1 or 2weeks of age reduces longevity and normalizes ("rescues") multiple aging-related traits. Collectively, these findings indicate that nutritional and hormonal signals during development can have profound impact on the trajectory of aging. We suspect that altered "programming" of aging during development may represent one of the mechanisms of the Developmental Origins of Health and Disease (DOHaD) and the detrimental effects of "catch-up" growth.

Effects of adult onset mild calorie restriction on weight of reproductive organs, plasma parameters and gene expression in male mice.

Calorie restriction (CR) extends lifespan and delays onset of age-related diseases in various organisms, even when started later in life. Despite benefits for health and lifespan, CR's negative impact on reproduction is documented in some animals. Studies employing approximately 40% CR detected a delay in sexual maturation and impairment of fertility, which were combined with extension of the reproductive period. In contrast, mild CR (10-20%) is apparently not deleterious to reproduction. Hence, we hypothesized that mild CR started at 8 months of age would prolong reproductive capabilities and improve health parameters of male mice. To test this hypothesis, we assessed the effects of 10 and 20% CR on reproductive organ weights, selected plasma parameters and hepatic/testicular gene expression in normal male mice of heterogeneous genetic background. Starting at 8 months of age (adult), mice were assigned to 3 regimen groups: 10% CR (n = 8), 20% CR (n = 9) or ad libitum (AL; n = 8). Four months of CR were sufficient to reduce glycemia in a non-fasted protocol. Mild CR initiated in adulthood did not significantly impact final body weight, most of the analyzed plasma parameters or weight of androgen-dependent organs. Moreover, CR did not interfere with expression of the assessed testicular genes, or most of the hepatic genes, but it did cause an increase in the levels of peroxisome proliferator-activated receptor gamma (Pparg) and mouse sulfotransferase (mSTa); and a decrease in glucose-6-phosphatase-α (G6pc) mRNA, which might signify improvement of body condition. The important finding of our study was that a mild CR regimen, as low as 10 and 20%, was sufficient to impair glycemia in a non-fasted state, and also the levels of plasma IGF-1, corroborating the concept that mild CR has the potential for improving health and longevity, even when started later in life.

Prolonged exposure to GH impairs insulin signaling in the heart.

Acromegaly is associated with cardiac hypertrophy, which is believed to be a direct consequence of chronically elevated GH and IGF1. Given that insulin is important for cardiac growth and function, and considering that GH excess induces hyperinsulinemia, insulin resistance, and cardiac alterations, it is of interest to study insulin sensitivity in this tissue under chronic conditions of elevated GH. Transgenic mice overexpressing GH present cardiomegaly and perivascular and interstitial fibrosis in the heart. Mice received an insulin injection, the heart was removed after 2  min, and immunoblotting assays of tissue extracts were performed to evaluate the activation and abundance of insulin-signaling mediators. Insulin-induced tyrosine phosphorylation of the insulin receptor (IR) was conserved in transgenic mice, but the phosphorylation of IR substrate 1 (IRS1), its association with the regulatory subunit of the phosphatidylinositol 3-kinase (PI3K), and the phosphorylation of AKT were decreased. In addition, total content of the glucose transporter GLUT4 was reduced in transgenic mice. Insulin failed to induce the phosphorylation of the mammalian target of rapamycin (mTOR). However, transgenic mice displayed increased basal activation of the IR/IRS1/PI3K/AKT/mTOR and p38 signaling pathways along with higher serine phosphorylation of IRS1, which is recognized as an inhibitory modification. We conclude that GH-overexpressing mice exhibit basal activation of insulin signaling but decreased sensitivity to acute insulin stimulation at several signaling steps downstream of the IR in the heart. These alterations may be associated with the cardiac pathology observed in these animals.

Vitamin D3 cannot revert desensitization of growth hormone (GH)-induced STAT5-signaling in GH-overexpressing mice non-calcemic tissues.

Growth hormone (GH) binding to a membrane receptor dimer triggers multiple intracellular signaling pathways. Signal transducers and activators of transcription are the most relevant of these pathways for GH action. GH also activates several inhibitory mechanisms, particularly suppressors of cytokine signaling (SOCS/CIS) proteins. GH-overexpressing mice exhibit hepatic desensitization of the JAK2/STAT5 GH-signaling pathway, associated with an increased abundance of CIS. Vitamin D3 has been shown to inhibit GH-induced expression of CIS and SOCS-3 and therefore prolong GH signaling in osteoblast-like cells. The purpose of the present study is to determine if vitamin D3 could attenuate CIS expression in GH-overexpressing mice, and consequently allow GH JAK2/STAT5 signaling in GH-responsive tissues in these animals. The abundance of CIS, SOCS-2, SOCS-3, STAT5b and GHR, as well as STAT5b tyrosine phosphorylation after a GH stimulus, were measured in liver and muscle of GHRH-transgenic mice treated with 1alpha,25-dihydroxyvitamin D3 for 7 days. This treatment did not diminish CIS expression in GH-overexpressing mice tissues, nor did the content of SOCS-2 and SOCS-3 significantly vary. GH-induced STAT5b phosphorylation levels were similar to basal values in transgenic mice liver treated with or without vitamin D; the refractoriness to GH was also present in muscle. Therefore, treatment with vitamin D was not sufficient to revert STAT5 GH signaling desensitization in non-calcemic tissues in GH-overexpressing mice.

Differential regulation of membrane associated-growth hormone binding protein (MA-GHBP) and growth hormone receptor (GHR) expression by growth hormone (GH) in mouse liver.

Growth hormone (GH) binding to GH receptor (GHR) is the initial step that leads to the physiological functions of the hormone. Proteolytical cleavage of the GHR in humans and rabbits and alternative processing of the GHR transcript in rodents generates circulating growth hormone binding protein (GHBP). Moreover, other GHR truncated forms that result from alternative processing of the GHR mRNA transcript have been described. These GHR short forms are inserted in the plasma membrane but they are unable to transduce the signal. In rodents, membrane associated-GHBP (MA-GHBP), which accounts for a significant proportion of liver GH binding capacity, represents the main GHR short form found in membranes, and may therefore function as a negative form of the receptor. In the present study, GHR and MA-GHBP content in liver were analyzed using mutant and transgenic mice expressing different concentrations of growth hormone to evaluate the correlation between GH levels, body weight (BW), GHR and MA-GHBP expression. It was found that GH deficiency was associated with diminished BW, GHR and MA-GHBP expression, while increased GH concentration led to increased BW, GHR and MA-GHBP expression, but MA-GHBP upregulation was more pronounced than the observed increase in GHR expression. Since GHR and MA-GHBP both contribute to liver GH binding capacity, GH-induced enrichment of the dominant negative form would represent a compensatory mechanism triggered by high levels of the hormone. This attempt to attenuate the effects of supraphysiological concentrations of GH may be critical to reduce or prevent their plausible damaging effects on the organism.

Short-term caloric restriction does not modify the in vivo insulin signaling pathway leading to Akt activation in skeletal muscle of Ames dwarf (Prop1(df)/Prop1(df)) mice.

The purpose of this study was to analyze the interaction between caloric restriction (CR) and the dwarf mutation at the level of insulin sensitivity and signal transduction. To this end, we analyzed the in vivo status of the insulin signaling system in skeletal muscle from Ames dwarf (df/df) and normal mice fed ad libitum or subjected to short-term (20-day) CR. We measured insulin-stimulated phosphorylation of the IR and IRS-1, IRS-1-p85 association and Akt activation, and the abundance of the IR, IRS-1, p85, GLUT-4 and IGF-1 receptor in skeletal muscle. In terms of glucose homeostasis, the response to CR was different in both groups of animals. In normal animals, CR induced a significant reduction in both circulating insulin and glucose levels, while CR did not modify these parameters in df/df mice. We did not find any significant alteration in either activation or abundance of signaling molecules analyzed after short-term CR in either normal or Ames dwarf mice. We conclude that the initial adaptation to CR in normal mice is an increase in insulin sensitivity without changes in insulin signal transduction, and that this adaptation is not evidenced in df/df mice, probably since they are already hypersensitive to insulin.

Increased SH2-Bbeta content and membrane association in transgenic mice overexpressing GH.

Transgenic mice overexpressing GH present a marked GH signaling desensitization, reflected by low basal phosphorylation levels of the tyrosine kinase JAK2, and signal transducer and activator of transcription-5 (STAT5) and a lack of response of these proteins to a high GH dose. To evaluate the mechanisms involved in the regulation of JAK2 activity by high GH levels in vivo, the content and subcellular distribution of SH2-Bbeta were studied in GH-overexpressing transgenic mice. SH2-B is a member of a conserved family of adapter proteins characterized by the presence of a C-terminal SH2 domain, a central pleckstrin homology (PH) domain, and an N-terminal proline rich region. The isoform SH2-Bbeta modulates JAK2 activity by binding to the phosphorylated enzyme, further increasing its activity. However, it may also interact with non-phosphorylated inactive JAK2 via lower affinity binding sites, preventing abnormal activation of the kinase. SH2-Bbeta may also function as an adapter protein, acting as a GH signaling mediator. We now report that, in an animal model of GH excess in which JAK2 is not phosphorylated, although it is increased in the membrane-fraction, both the level of SH2-Bbeta, and especially its association to membranes, are augmented (67% and 13-fold vs normal mice values respectively), suggesting SH2-Bbeta could modulate JAK2 activity in vivo.

The impact of altered insulin-like growth factor-I secretion on the neuroendocrine and testicular functions.

There is unequivocal evidence that the biosynthesis and secretion of gonadotropins by the pituitary gland is controlled by the hypothalamic GnRH and the function of the testis is mainly regulated by FSH and LH. However, a number of investigations have suggested the role of other hormones/factors, including insulin-like growth factor-I (IGF-I) in the control of pituitary and gonadal functions. The role of growth hormone (GH) and IGF-I is poorly investigated in humans. In animals with altered IGF-I secretion, the gonadotropin and androgen secretions are affected. Similarly, there is evidence that fertility, the onset of puberty and sexual maturation are affected in some patients with Laron syndrome and in acromegaly. In this minireview, we have presented some data obtained in humans and also included results from several experimental models with altered GH/IGF-I secretion, in the hope that the results from animals will possibly help in understanding the important role of IGF-I in the control of neuroendocrine-testicular function in humans.

Increased sensitivity to GH in liver of Ames dwarf (Prop1df/Prop1df) mice related to diminished CIS abundance.

To investigate the influence of chronic GH deficiency on GH signaling in vivo, we have analyzed Janus kinase (JAK) 2/signal transducers and activators of transcription (STAT) 5 GH signaling pathway, and its regulation by the suppressors of the cytokine signaling SOCS and by the JAK2-interacting protein SH2-Bbeta, in liver of Ames dwarf (Prop1df/Prop1df) mice, which are severely deficient in GH, prolactin and TSH, and of their normal littermates. Prop1df/Prop1df mice displayed unaltered GH receptor, JAK2 and STAT5a/b protein levels. No significant differences in the basal tyrosine-phosphorylation levels of JAK2 and STAT5a/b were found between both groups of animals. After in vivo administration of a high GH dose (5 microg/g body weight (BW)), the tyrosine-phosphorylation levels of JAK2 and STAT5a/b increased significantly, reaching similar values in normal and dwarf mice. However, after stimulation with lower GH doses (50 and 15 ng/g BW) the tyrosine-phosphorylation level of STAT5a/b was higher in dwarf mice. The protein content of CIS, a SOCS protein that inhibits STAT5 signaling, was approximately 80% lower in dwarf mice liver, while SOCS-2 and SOCS-3 levels were unaltered. The content of SH2-Bbeta, a modulator of JAK2 activity, was reduced by approximately 30% in dwarf mice, although this was associated with normal JAK2 response to a high GH dose. In summary, Prop1df/Prop1df mice display increased hepatic sensitivity to GH, an effect that could be related to the lower abundance of CIS in this tissue. Furthermore, the lower CIS content found in this model of GH deficiency suggests that CIS protein levels are regulated by GH in vivo.

Effects of Soy-derived diets on plasma and liver lipids, glucose tolerance, and longevity in normal, long-lived and short-lived mice.

We examined the effects of diets based on a low isoflavone or a high isoflavone soy protein isolates in normal, growth-hormone receptor knockout and Ames dwarf, and Prop 1 (df) mice that are hypoinsulinemic, insulin-sensitive, and exceptionally long-lived, as well as in growth hormone transgenic mice that are hyperinsulinemic, insulin-resistant, dyslipidemic, and short-lived. Soybean diets tended to normalize plasma cholesterol levels in dwarf and transgenic mice, while low isoflavone diet reduced plasma triglycerides in most of the examined genotypes. The effects of low isoflavone and high isoflavone diets on the levels of free and esterified cholesterol in the liver were strongly genotype-dependent. Fasting blood glucose levels were reduced and glucose tolerance improved by both low isoflavone and high isoflavone diets in growth hormone-transgenic mice and in their normal siblings. Glucose tolerance was also improved by high-isoflavone diet in growth hormone receptor knockout mice. Lifespan was increased by low isoflavone diet in normal mice from two of the examined stocks. High isoflavone diet increased lifespan in normal animals from one line, but reduced lifespan of normal mice from a different line. We conclude that dietary soy protein intake can improve plasma and hepatic lipid profiles, reduce fasting glucose, enhance capacity for glucose tolerance, and prolong life, but all of these effects are strongly genotype-dependent.

Differential effects of light/dark recombinant human prolactin administration on the submaxillary lymph nodes and spleen activity of adult male mice.

OBJECTIVES: Day/night variations in cellularity, percentage of CD4+, CD8+ and double-positive (CD4+-CD8+) lymphocytes, lipopolysaccharide (LPS)- and concanavalin A (Con A)-induced lymphocyte proliferation and natural killer (NK) activity, and the effect of timed administration of recombinant human prolactin (h-PRL) on the above-mentioned parameters were investigated in the submaxillary lymph nodes and spleen of adult male mice. RESULTS: In controls, the percentage of CD4+, double-positive lymphocytes, LPS- or Con A-induced blastogenic proliferation and NK activity in the spleen differ during the dark phase as compared to the light phase. When administered during the dark period, h-PRL induced immunosuppresion in the percentage of CD4+, double-positive (CD4+-CD8+) lymphocytes. Con A- and LPS-induced lymphocyte proliferation and NK activity as compared to untreated controls. When h-PRL was administered during the light period, the cellularity increased, and h-PRL was immunosuppressive in Con A- and LPS-induced lymphcoyte proliferation and NK activity as compared to controls. Moreover, in control submaxillary lymph nodes the cellularity, percentage of CD8+, double-positive lymphocytes, blastogenic proliferation in the presence of Con A and LPS and NK activity differ when comparing the dark with the light phase. When administered during the dark period h-PRL induced immunosuppression in the percentage of double-positive (CD4+-CD8+) lymphocytes, Con A- and LPS-induced lymphocyte proliferation as compared to controls. When h-PRL is administered during the light period, no effects were observed. CONCLUSIONS: These results indicate the existence of differential day/night variations in the cellular immune response depending upon the lymphoid organ considered. Because of the administration of h-PRL a differential modulation of this circadian variation was also observed.

Could a deficiency in growth hormone signaling be beneficial to the aging brain?

Several studies have shown that growth hormone (GH)-deficient/resistant animals have a prolonged lifespan compared with their normal siblings. Studies in our laboratory have suggested that both Ames dwarf and GH receptor/GH binding protein knockout (GH-R-KO) mice do not experience age-induced cognitive aging at the same rate as their normal siblings. The studies presented here were aimed at determining whether these long-lived mice experience a delay in age-related changes in behavior. Young and old mice of both strains were tested in an open-field task. In addition, mice of the GH-R-KO strain were tested in the water maze to confirm previous findings using the inhibitory avoidance task that suggested delayed cognitive aging. In each of these studies, normal (wild-type) animals of the same age, sex, and genetic background as the mutants served as controls. Old GH-R-KO mice did not experience the decline in locomotor activity or difference in activity levels in the open-field task seen in the normal animals. Young normal and young and old Ames dwarf mice spent less time in the center of the apparatus compared with old normal animals. There were no signs of age-related changes in emotionality within the GH-R-KO strain. Water maze results also showed that while old normal animals performed poorer than the young normal animals, old GH-R-KO mice did not perform differently from the young normal or young GH-R-KO groups. Taken together, these studies support our previous findings of delayed age-induced cognitive and behavioral decline in GH deficient/resistant mice.

Insulin-like growth factor 1 (IGF-1) and aging: controversies and new insights.

Insulin/insulin-like growth factor (IGF) signaling plays a major role in the control of aging and life span in invertebrates. Major extension of life span in growth hormone receptor knock out (GHR-KO) mice that are GH resistant, and subsequently, IGF-I-deficient indicates that similar mechanisms may operate in mammals. This conclusion is supported by association of reduced IGF-I levels and delayed aging in three different GH-deficient mutant mice and in animals subjected to caloric restriction, but is difficult to reconcile with neuroprotective effects of IGF-I and with the suspected role of declining GH levels during aging. We suggest that the role of IGF in the regulation of growth and adult body size is important in mediating the effects of longevity genes on aging and life span. Suspected mechanisms of IGF-I action in aging also include reduced insulin signaling, enhanced sensitivity to insulin, and reduced thermogenesis with diminished oxidative damage of macromolecules being the likely final common pathway of these effects. We suspect that IGF-I is important in evolutionarily conserved mechanisms that link life history, including development, reproduction, and aging with availability of energy resources.

Antioxidant enzymes, free-radical damage, and response to paraquat in liver and kidney of long-living growth hormone receptor/binding protein gene-disrupted mice.

Numerous studies have shown that the lifespan can be extended by caloric restriction or by altering the growth hormone (GH)-insulin-like growth factor 1 signaling pathway. Both of these manipulations produce physiological alterations, such as increased insulin sensitivity, and reduced glucose levels and body size. However, it is difficult to evaluate whether these are merely correlates of delayed aging or whether they have a direct causal effect on lifespan. One parameter that has been demonstrated to have causal, positive effects on longevity in invertebrates is improved antioxidant defenses. We measured activities of antioxidant enzymes Cu/Zn superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GPx) and quantified free-radical damage by lipid peroxidation (LP) and protein oxidation (PO) measurements in liver and kidney tissues, and evaluated the response to paraquat-induced oxygen toxicity in the long-living GH receptor/binding protein gene knockout (GHR-KO) mouse. We found that in the kidney, SOD was lower and GPx was higher in GHR-KO mice, and LP was higher in female GHR-KO mice only. In the liver, female GHR-KO mice had lower GPx, while male GHR-KO mice had lower CAT and higher LP. GHR-KO males were also more susceptible to paraquat toxicity compared to females or normal males. We conclude that in long-living GHR-KO mice, GH-resistance does not confer longevity by improved free-radical scavenging in the liver and kidney, suggesting that greater free-radical defenses in other tissues, or altered glucose metabolism may have a more central role in extending the lifespan of these animals.

Consequences of growth hormone (GH) overexpression and GH resistance.

Development of transgenic mice overexpressing GH and GHR-KO mice with GH resistance provided novel animal models for study of the somatotropic axis and for identifying GH actions that may be relevant to its current and contemplated use in medicine and agriculture. Studies of phenotypic characteristics of these animals revealed previously unsuspected actions of GH and IGF-I on neuroendocrine functions related to reproduction and to the release of "stress hormones" (glucocorticoids and prolactin). These studies also provided novel and still-disputed evidence for involvement of somatotropic axis in the control of aging and life span and in mediating the actions of longevity genes.