Mature Adult Mice Research Articles

Acute brain slice methods for adult and aging animals: application of targeted patch clamp analysis and optogenetics.

The development of the living acute brain slice preparation for analyzing synaptic function roughly a half century ago was a pivotal achievement that greatly influenced the landscape of modern neuroscience. Indeed, many neuroscientists regard brain slices as the gold-standard model system for detailed cellular, molecular, and circuitry level analysis and perturbation of neuronal function. A critical limitation of this model system is the difficulty in preparing slices from adult and aging animals, and over the past several decades few substantial methodological improvements have emerged to facilitate patch clamp analysis in the mature adult stage. In this chapter we describe a robust and practical protocol for preparing brain slices from mature adult mice that are suitable for patch clamp analysis. This method reduces swelling and damage in superficial layers of the slices and improves the success rate for targeted patch clamp recordings, including recordings from fluorescently labeled populations in slices derived from transgenic mice. This adult brain slice method is suitable for diverse experimental applications, including both monitoring and manipulating neuronal activity with genetically encoded calcium indicators and optogenetic actuators, respectively. We describe the application of this adult brain slice platform and associated methods for screening kinetic properties of Channelrhodopsin (ChR) variants expressed in genetically defined neuronal subtypes.

PERK Is Required in the Adult Pancreas and Is Essential for Maintenance of Glucose Homeostasis

Germ line PERK mutations are associated with diabetes mellitus and growth retardation in both rodents and humans. In contrast, late embryonic excision of PERK permits islet development and was found to prevent onset of diabetes, suggesting that PERK may be dispensable in the adult pancreas. To definitively establish the functional role of PERK in adult pancreata, we generated mice harboring a conditional PERK allele in which excision is regulated by tamoxifen administration. Deletion of PERK in either young adult or mature adult mice resulted in hyperglycemia associated with loss of islet and β cell architecture. PERK excision triggered intracellular accumulation of proinsulin and Glut2, massive endoplasmic reticulum (ER) expansion, and compensatory activation of the remaining unfolded-protein response (UPR) signaling pathways specifically in pancreatic tissue. Although PERK excision increased β cell death, this was not a result of decreased proliferation as previously reported. In contrast, a significant and specific increase in β cell proliferation was observed, a result reflecting increased cyclin D1 accumulation. This work demonstrates that contrary to expectations, PERK is required for secretory homeostasis and β cell survival in adult mice.

Organotypic Brain Slice Cultures of Adult Transgenic P301S Mice—A Model for Tauopathy Studies

BackgroundOrganotypic brain slice cultures represent an excellent compromise between single cell cultures and complete animal studies, in this way replacing and reducing the number of animal experiments. Organotypic brain slices are widely applied to model neuronal development and regeneration as well as neuronal pathology concerning stroke, epilepsy and Alzheimer’s disease (AD). AD is characterized by two protein alterations, namely tau hyperphosphorylation and excessive amyloid β deposition, both causing microglia and astrocyte activation. Deposits of hyperphosphorylated tau, called neurofibrillary tangles (NFTs), surrounded by activated glia are modeled in transgenic mice, e.g. the tauopathy model P301S.Methodology/Principal FindingsIn this study we explore the benefits and limitations of organotypic brain slice cultures made of mature adult transgenic mice as a potential model system for the multifactorial phenotype of AD. First, neonatal (P1) and adult organotypic brain slice cultures from 7- to 10-month-old transgenic P301S mice have been compared with regard to vitality, which was monitored with the lactate dehydrogenase (LDH)- and the MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assays over 15 days. Neonatal slices displayed a constant high vitality level, while the vitality of adult slice cultures decreased significantly upon cultivation. Various preparation and cultivation conditions were tested to augment the vitality of adult slices and improvements were achieved with a reduced slice thickness, a mild hypothermic cultivation temperature and a cultivation CO2 concentration of 5%. Furthermore, we present a substantial immunohistochemical characterization analyzing the morphology of neurons, astrocytes and microglia in comparison to neonatal tissue.Conclusion/SignificanceUntil now only adolescent animals with a maximum age of two months have been used to prepare organotypic brain slices. The current study provides evidence that adult organotypic brain slice cultures from 7- to 10-month-old mice independently of the transgenic modification undergo slow programmed cell death, caused by a dysfunction of the neuronal repair systems.

Age-related changes to Na+ channel gating contribute to modified intrinsic neuronal excitability

Cognitive decline occurs during normal aging and is likely to be reflected in the neurophysiological properties of neural circuits with key roles in cognition, for example those of the limbic system. To identify candidate neurophysiological changes we used patch clamp methods to compare the intrinsic excitability properties of hippocampal CA1 pyramidal neurons of mature adult (8–10 month) and aged (22–24 month) mice. Resting potential, input resistance, and the “sag” observed on injection of hyperpolarizing current were not age-dependent. In contrast, the patterns of spike firing observed with depolarizing current injections demonstrated the presence of an age-related hypoexcitability. Action potential waveform analysis revealed that spike thresholds were approximately 3 mV more depolarized in aged animals. In line with this, voltage clamp recordings of Na+ currents from nucleated macropatches exhibited an approximate 3 mV depolarizing shift in the voltage-dependence of activation gating. Inactivation curves, in contrast, were not different. These data indicate alterations in Na+ channel activation gating contribute to neuronal hypoexcitability in aging, and therefore may be a factor in age-related cognitive decline.

Impact of Unplanned Disposal of Tanneries Effluents on Testes – A Dilemma of Our Society

The modern industrialized society faces serious prob-lems of exposure of its population to the environmen-tal pollutants. Rapid industrialization is beneficial to mankind but at the same time it is creating problems due to unplanned and unwise disposal of industrial eff-luents particularly from the tanneries in the environ-ment. The wastes of industries utilizing the chromium as tanneries have substantial amount of chromium in it. This study was designed to analyze the effects of hexavalent chromium on diameter of seminiferous tub-ules of testes of mice at Animal House of Zoology Department, University of the Punjab, Lahore. This experiment was performed on forty – eight mature adult male albino mice, which were divided into eight groups. Control groups were designated as A 1 , B 1 , C 1 and D 1 , whereas experimental groups A, B, C and D, received chromium (2 mg / kg), i.p., as potassium di-chromate on alternate days for two, four, six and eight weeks respectively. Significant histological changes were recorded in the testes of treated mice, after pro-longed exposure of chromium tubular diameter of the seminiferous tubules was reduced. In case of chronic exposure more drastic deleterious effects will appear. Future research work will add further information in this regard. Key Words: Potassium dichromate, Testes, Semini-ferous tubules, hexavalent chromium.

Glial molecular alterations with mouse brain development and aging: up-regulation of the Kir4.1 and aquaporin-4

Glial cells, besides participating as passive supporting matrix, are also proposed to be involved in the optimization of the interstitial space for synaptic transmission by tight control of ionic and water homeostasis. In adult mouse brain, inwardly rectifying K+ (Kir4.1) and aquaporin-4 (AQP4) channels localize to astroglial endfeets in contact with brain microvessels and glutamate synapses, optimizing clearance of extracellular K(+) and water from the synaptic layers. However, it is still unclear whether there is an age-dependent difference in the expressions of Kir4.1 and AQP4 channels specifically during postnatal development and aging when various marked changes occur in brain and if these changes region specific. RT-PCR and immunoblotting was conducted to compare the relative expression of Kir4.1 and AQP4 mRNA and protein in the early and mature postnatal (0-, 15-, 45-day), adult (20-week), and old age (70-week) mice cerebral and cerebellar cortices. Expressions of Kir4.1 and AQP4 mRNA and protein are very low at 0-day. A pronounced and continuous increase was observed by mature postnatal ages (15-, 45-days). However, in the 70-week-old mice, expressions are significantly up-regulated as compared to 20-week-old mice. Both genes follow the same age-related pattern in both cerebral and cerebellar cortices. The time course and expression pattern suggests that Kir4.1 and AQP4 channels may play an important role in brain K(+) and water homeostasis in early postnatal weeks after birth and during aging.

Mature Adult Dystrophic Mouse Muscle Environment Does Not Impede Efficient Engrafted Satellite Cell Regeneration and Self-Renewal

Changes that occur in the skeletal muscle environment with the progress of muscular dystrophies may affect stem cell function and result in impaired muscle regeneration. It has previously been suggested that the success of stem cell transplantation could therefore be dependent both on the properties of the cell itself and on the host muscle environment. Here we engrafted young and mature adult mdx-nude mice, which are the genetic homolog of Duchenne muscular dystrophy, with a small number of satellite cells freshly isolated from young, normal donor mice. We found that the donor satellite cells contributed to muscle regeneration and self-renewal as efficiently within mature adult, as in young, dystrophic host muscle. Donor-derived satellite cells also contributed to robust regeneration after further injury, showing that they were functional despite the more advanced dystrophic muscle environment. These findings provide evidence that muscle tissue in a later stage of dystrophy may be effectively treated by stem cells.

Pregnancy in the mature adult mouse does not alter the proportion of mammary epithelial stem/progenitor cells

IntroductionIn humans, an early full-term pregnancy reduces lifetime breast cancer risk by up to 50% whereas a later pregnancy (>35 years old) can increase lifetime risk. Several mechanisms have been suggested, including changes in levels of circulating hormones, changes in the way the breast responds to these hormones, changes in gene expression programmes which may alter susceptibility to transformation and changes to mammary stem cell numbers or behaviour. Previous studies have shown that the mammary tissue isolated from both virgin and parous mice has the ability to repopulate a cleared mammary fat pad in transplant experiments. Limited dilution transplant assays have demonstrated that early pregnancy (at 5 weeks of age) reduces stem/progenitor cell numbers in the mouse mammary epithelium by twofold. However, the effects on stem/progenitor cell numbers in the mammary epithelium of a pregnancy in older animals have not yet been tested.MethodsMice were put through a full-term pregnancy at 9 weeks of age, when the mammary epithelium is mature. The total mammary epithelium was purified from parous 7-week post-lactation and age-matched virgin mice and analysed by flow cytometry and limiting dilution cleared fat pad transplants.ResultsThere were no significant differences in the proportions of different mammary epithelial cell populations or numbers of CD24+/Low Sca-1- CD49fHigh cells (stem cell enriched basal mammary epithelial compartment). There was no significant difference in stem/progenitor cell frequency based on limiting dilution transplants between the parous and age-matched virgin epithelium.ConclusionsAlthough differences between parous and virgin mammary epithelium at later time points post lactation or following multiple pregnancies cannot be ruled out, there are no differences in stem/progenitor cell numbers between mammary epithelium isolated from parous animals which were mated at 9 weeks old and virgin animals. However, a recent report has suggested that animals that were mated at 5 weeks old have a twofold reduction in stem/progenitor cell numbers. This is of interest given the association between early, but not late, pregnancy and breast cancer risk reduction in humans. However, a mechanistic connection between stem cell numbers and breast cancer risk remains to be established.

Adaptive β-Cell Proliferation Is Severely Restricted With Advanced Age

OBJECTIVERegeneration of the insulin-secreting β-cells is a fundamental research goal that could benefit patients with either type 1 or type 2 diabetes. β-Cell proliferation can be acutely stimulated by a variety of stimuli in young rodents. However, it is unknown whether this adaptive β-cell regeneration capacity is retained into old age.RESEARCH DESIGN AND METHODSWe assessed adaptive β-cell proliferation capacity in adult mice across a wide range of ages with a variety of stimuli: partial pancreatectomy, low-dose administration of the β-cell toxin streptozotocin, and exendin-4, a glucagon-like peptide 1 (GLP-1) agonist. β-Cell proliferation was measured by administration of 5-bromo-2′-deoxyuridine (BrdU) in the drinking water.RESULTSBasal β-cell proliferation was severely decreased with advanced age. Partial pancreatectomy greatly stimulated β-cell proliferation in young mice but failed to increase β-cell replication in old mice. Streptozotocin stimulated β-cell replication in young mice but had little effect in old mice. Moreover, administration of GLP-1 agonist exendin-4 stimulated β-cell proliferation in young but not in old mice. Surprisingly, adaptive β-cell proliferation capacity was minimal after 12 months of age, which is early middle age for the adult mouse life span.CONCLUSIONSAdaptive β-cell proliferation is severely restricted with advanced age in mice, whether stimulated by partial pancreatectomy, low-dose streptozotocin, or exendin-4. Thus, β-cells in middle-aged mice appear to be largely postmitotic. Young rodents may not faithfully model the regenerative capacity of β-cells in mature adult mice.

The PPARγ agonist FMOC- l-leucine protects both mature and immature brain

( N-[9-fluorenylmethoxycarbonyl]-)- l-leucine (FMOC- l-leucine) and rosiglitazone, two ligands of peroxisome proliferator-activated receptor γ (PPARγ), were evaluated in mature (adult mice) and immature (pups) brain injury models. In adult magnesium-deficient mice, a model responsive to both neuroprotective and anti-seizure compounds, FMOC- l-leucine, but not rosiglitazone, protected against audiogenic seizures. The protection afforded by FMOC- l-leucine was alleviated by the PPARγ antagonist GW9662 (1–2 mg/kg) and was induced in 50% animals by 4.8 ± 1.2 mg/kg. At this dose, FMOC- l-leucine modified audiogenic seizure phase durations in convulsing mice differently than prototype antiepileptic drugs did. FMOC- l-leucine (up to 100 mg/kg) was inactive in the 6 Hz seizure test, an adult animal model largely responsive to anti-seizure drugs. In a model of neonatal brain injury, FMOC- l-leucine (4 μg/kg) was neuroprotective against cerebral ibotenate toxicity. It reduced significantly the size of lesions in grey but not in white matter, while rosiglitazone (10 μg/kg) was inactive. Taken as a whole, the present data support neuroprotective potentialities of FMOC- l-leucine towards both mature and immature brain. The PPAR-based protection of immature brain is more important as it is known that classic adult brain protectants (GABA A activators, N-methyl- d-aspartate and sodium channel blockers) may be toxic for immature brain. The PPARγ agonist FMOC- l-leucine is likely to be devoid of these classic protective mechanisms because of its inactivity in the 6 Hz seizure test, its activity in the audiogenic test being explained by neuroprotective rather than intrinsic anti-seizure mechanisms. Targeting PPARs might be thus a promising way to protect immature brain.

AGE-DEPENDENT RESPONSES TO HEPATIC ISCHEMIA/REPERFUSION INJURY

The current study explored the concept that adult and pediatric populations differ in their response to major injury. Male C57BL/6 mice of a "young adult" (8-12 weeks) or "mature adult" (12-13 months) age were subjected to partial hepatic ischemia and reperfusion. Mature adult mice displayed significantly more liver injury than young adult mice as assessed histologically and by serum levels of alanine aminotransferase. Interestingly, there was far less neutrophil accumulation in the livers of mature adult mice. However, liver-recruited neutrophils from mature adult mice had a higher activation state than those from young adult mice. Activation of the inflammatory transcription factor, NF-kappaB, was suppressed in whole livers from mature adult mice. In isolated liver cells, Kupffer cells showed no difference in NF-kappaB activation, but hepatocytes from mature adult mice had delayed NF-kappaB activation in response to TNF. Furthermore, isolated hepatocytes from young adult mice produced abundant amounts of the chemokine, macrophage inflammatory protein-2, whereas hepatocytes from mature adult mice produced little, if any macrophage inflammatory protein-2. Mature adult mice had much lower hepatic expression of the cytoprotective protein, heat shock protein 70, than did young adult mice. In contrast, serum heat shock protein 70 levels, which has been linked to subsequent tissue injury, were higher in mature adult mice than in young adult mice. These data suggest that there are multiple alterations at the cellular and molecular levels that contribute to enhanced postischemic liver injury in mature adult mice.

Gonadotropin-releasing hormone regulates expression of the DNA damage repair gene, Fanconi anemia A, in pituitary gonadotroph cells.

Gonadal function is critically dependant on regulated secretion of the gonadotropin hormones from anterior pituitary gonadotroph cells. Gonadotropin biosynthesis and release is triggered by the binding of hypothalamic GnRH to GnRH receptor expressed on the gonadotroph cell surface. The repertoire of regulatory molecules involved in this process are still being defined. We used the mouse L beta T2 gonadotroph cell line, which expresses both gonadotropin hormones, as a model to investigate GnRH regulation of gene expression and differential display reverse transcription-polymerase chain reaction (RT-PCR) to identify and isolate hormonally induced changes. This approach identified Fanconi anemia a (Fanca), a gene implicated in DNA damage repair, as a differentially expressed transcript. Mutations in Fanca account for the majority of cases of Fanconi anemia (FA), a recessively inherited disease identified by congenital defects, bone marrow failure, infertility, and cancer susceptibility. We confirmed expression and hormonal regulation of Fanca mRNA by quantitative RT-PCR, which showed that GnRH induced a rapid, transient increase in Fanca mRNA. Fanca protein was also acutely upregulated after GnRH treatment of L beta T2 cells. In addition, Fanca gene expression was confined to mature pituitary gonadotrophs and adult mouse pituitary and was not expressed in the immature alpha T3-1 gonadotroph cell line. Thus, this study extends the expression profile of Fanca into a highly specialized endocrine cell and demonstrates hormonal regulation of expression of the Fanca locus. We suggest that this regulatory mechanism may have a crucial role in the GnRH-response mechanism of mature gonadotrophs and perhaps the etiology of FA.

Exposure of mice to a predator odor increases acoustic startle but does not disrupt the rewarding properties of VTA intracranial self-stimulation

The present investigation assessed the propensity of an acute psychogenic stressor exposure to induce behavioral change in paradigms assessing fear/anxiety (acoustic startle) and motivation/anhedonia (intracranial self-stimulation) in CD-1 mice. In the acoustic startle paradigm, a 10-min exposure of 2–4 month old mice (young adult mice) to fox odor (2,5-dihydro-2,4,5-trimethylthiazoline; TMT) was associated with decreased acoustic startle relative to mice exposed to the control odor, butyric acid (BA), immediately and relative to both saline and BA exposure 24 h following odor exposure in the home cage. In contrast, a 2-min exposure of young adult mice to TMT was associated with an increase in startle relative to saline and BA during the immediate post-odor test session only. In young adult mice a 2-min and a 10-min exposure to BA resulted in a startle profile of mice reminiscent of saline-treated mice. In comparison to young adult mice, a 2-min exposure of mature adult mice (5–7 months old) to TMT enhanced startle for up to 48 h relative to both saline and BA, while a 10-min exposure of mature adult mice to TMT enhanced startle for 168 h post-odor exposure relative to saline-exposed mice only. However, the greatest increase in startle amplitude (i.e. 48 h) was acquired following the 2-min exposure of mature mice to TMT. Among mature adult mice, a 10-min exposure to BA in the home cage eventuated in enhanced startle relative to saline-exposed animals 168 h following odor exposure. In comparison, exposure of mice to 10 min of TMT depressed responding for VTA brain stimulation at the initial 80 Hz frequency, but was ineffective in elevating reward thresholds relative to mice merely exposed to saline. Mice assessed in the ICSS paradigm were approximately 2–4 months old at the time of surgery and 5–7 months old at the completion of testing. These data suggest that acute odor exposure may induce a fear gradient dependent upon the perceived stressor severity and that the resultant anxiety-like effects are dependent on the duration of odor exposure, age of the animals and the temporal interval between odor presentation and behavioral testing. Moreover, the anxiogenic properties of psychogenic stressors can be separated from their anhedonic effects. The implications of these data for clinical psychopathology are discussed.

Allogeneic offspring produced by male germ line stem cell transplantation into infertile mouse testis.

The testis is one of several immune-privileged organs and is known for its unique ability to support allogeneic or xenogeneic tissue transplants. We investigated the possibility of deriving offspring from mice that underwent transplantation with allogeneic male germ line stem cells in the testis. Although mature adult mice rejected allogeneic germ cells and were infertile, offspring were obtained by intracytoplasmic germ cell injection using partially differentiated donor cells. In contrast, complete spermatogenesis occurred when allogeneic germ cells were transplanted into immature pup testes. Tolerance induction by monoclonal antibody administration allowed the pup transplant recipients to produce allogeneic offspring by natural mating, whereas no spermatozoa were found in the epididymis of untreated recipients. Thus, these results indicate that a histoincompatible recipient can serve as a "surrogate father" to propagate the genetic information of heterologous male donors.

Expression and immune recognition of SV40 Tag in transgenic mice that develop metastatic osteosarcomas.

Mature adult mice of the C57BL/6-TgN(Amy1TAg)501Knw transgenic mouse lineage, 501, containing a liver alpha-amylase promoted-SV40 Tag hybrid gene, routinely develop SV40 Tag-induced metastatic osteosarcomas. This form of alpha-amylase was known to be expressed in the liver, salivary glands, pancreas, and fat. Cells in the normal rib adjacent to the periosteum also express alpha-amylase suggesting that transgene expression is correctly targeted to generate osteosarcomas. 501 mice express SV40 Tag in the salivary glands but do not develop abnormalities in these organs by the time of their death from SV40-induced osteosarcomas. Mice of the C57BL/6 strain make a strong and effective anti-tumor immune response to SV40 Tag immunization. However, immunization of 501 mice with SV40 Tag early in life does not alter or prevent SV40 Tag-induced osteosarcomagenesis. 501 mice mount a significantly less effective cytotoxic T-lymphocyte response following SV40 Tag immunization while 501 osteosarcoma-derived cells are fully susceptible to SV40 Tag-specific T-cell lysis. This suggests that partial tolerance, not loss of antigen presentation by tumor cells, characterizes this mouse model of endogenous bone tumor development. To determine whether the immune recognition of endogenous SV40 Tag could influence tumorigenesis, the metastatic potential and time of death from tumor was investigated in CD4-null mutant 501 mice and beta-2 microglobulin-null mutant 501 mice. The size and number of metastases in these strains and longevity of these strains varied. We suggest that components of both the innate and adaptive immune response control tumor appearance and progression.

Early postnatal cocaine exposure causes sequential, dose-dependent, enduring but reversible supersensitivity in 5-HT 2A receptor-mediated function during development in male mice

This report investigated whether postnatal exposure to cocaine affects the index of 5-HT 2A receptor function during development by utilizing the ability of the 5-HT 2A/C agonist DOI to induce the head-twitch response (HTR) in mice. Thus, several groups of mice litters were treated with varying doses of cocaine (0, 0.5, 1, 5, and 10 mg/kg, IP) twice daily from postnatal days 5 to 14. Then, different groups of cocaine-exposed male mice pups along with their corresponding age-matched vehicle-exposed control groups were HTR tested once during development on the following postnatal test days: 15, 16, 18, 20, 30, 45, and 60. The HTR testing involved administration of DOI (0.5 mg/kg, IP) and counting the frequency of the behavior for the next 20 min. Cocaine exposure caused bell-shaped, dose-dependent, enduring but reversible increase in DOI-induced HTR frequency (mean ± SEM) during development. The developing pups were most sensitive to low and intermediate doses of cocaine (0.5–5 mg/kg). The greatest degree of increase in HTR frequency in response to DOI challenge occurred in the 1 mg/kg cocaine-exposure group on most test days. The onset of HTR supersensitivity varied from 48 h (5 mg/kg) to 144 h (0.5 mg/kg) following the termination of chronic cocaine exposure. Moreover, maximal supersensitivity for the latter doses of cocaine occurred 96 and 384 h postcocaine treatment, respectively. Other cocaine exposure groups attained their maxima sometime between the latter time periods. The duration of persistence of 5-HT 2A receptor supersensitivity varied with different doses of cocaine: the 10-mg/kg group was supersensitive up to 384 h postcocaine treatment, the 1- and 5-mg/kg groups up to 744 h; and the 0.5-mg/kg group up to 1104 h. Although developmentally cocaine-exposed pups exhibit some similarities (i.e., exquisite sensitivity and bell-shaped dose–response) in 5-HT 2A receptor adaptation to mature adult mice exposed to cocaine, they also differ from mature adult cocaine-exposed mice in the onset of appearance as well as the enduring persistence of the induced supersensitivity.

The induction of systemic and mucosal immune responses following the subcutaneous immunization of mature adult mice: characterization of the antibodies in mucosal secretions of animals immunized with antigen formulations containing a vitamin D3 adjuvant

Systemic and mucosal immune responses were effectively induced following the subcutaneous administration of Haemophilus influenzae type b oligosaccharide conjugated to diphtheria toxoid vaccine in a formulation containing the active form of vitamin D3. IgA and IgG antibodies with specificity for both the protein and oligosaccharide components of the vaccine were detectable in mucosal secretions following immunization. The IgA and IgG mucosal antibodies were produced locally, and were functional as demonstrated by their diphtheria toxin neutralizing activity. Our data suggests that subcutaneous tissues can effectively serve as effective antigen presenting sites for both mucosal and systemic immune responses to antigens administered in combination with vitamin D3.

Importance of estrogens in human males' fertility and bone pathophysiology

Since estrogens play a crucial role in several processes such as embryo implantation and fetal development it has been thought that mutation of the estrogenreceptor (ER) gene would be lethal. This hypothesis was supported by the fact that no mutations of the ER gene leading to estrogen insensitivity had been observed in mammalians. In contrast, conditions of resistance to other hormones (tri-iodothyronine, vitamin D, cortisol, androgens), which also act via receptors belonging to the nuclear receptor superfamily, have been reported with increasing frequency. However, this notion changed in 1994 when Korach (1) created a mutant mouse line without a functional ER, obtained by the gene knock-out technique (ER knock-out; ERKO). These animals provided an attractive experimental model which could be used to gain a better understanding of the role of estrogens in a variety of tissues during early developmental stages. The main finding was that crosses of the heterozygous ERKO mice resulted in live birth offspring containing the expected Mendelian distribution of the three possible genotypes. This result suggested that, in contrast to what was previously thought, disruption of the ER was not crucial for survival. Furthermore, the homozygous ERKO mice showed no bias in sex ratio, indicating that sex determination was not affected by the absence of the estrogen receptor. As far as the phenotype was concerned, both sexes of the homozygous ERKO mice were infertile, as demonstrated by mating studies, and exhibited a variety of changes, some of which were associated with the gonads, mammary glands, reproductive tracts and skeletal tissues. In particular, homozygous mutant female mice showed the presence of hypoplastic reproductive tract structures, hemorrhagic cystic ovaries with no functional corpora lutea and no uterine response to estrogen treatment. The mammary glands were undeveloped with only vestigial ducts present at the nipples. Of particular relevance was the observation that the bone density of the ERKO male and female mice was 20 to 25% lower than in wild-type mice, suggesting a direct role for estrogen in bone physiology. An unexpected finding was the infertility of the sexually mature male adult ERKO mice, despite the presence of anatomically normal reproductive apparatus. In order to assess the essential role for ER-mediated processes in the regulation of male reproduction, further studies on ERKO male mice were recently carried out by Eddy and colleagues (2). These authors demonstrated that the absence of ER was detrimental to spermatogenesis, sperm function and mating performance. Although the findings in ERKO mice were exciting, their relevance to human physiology appeared to be questionable, due to the species differences in genetic background. Nevertheless, the first case report of estrogen resistance in man due to mutation in the ER gene (3) provided the evidence that the ERKO mouse could be an acceptable model for the evaluation of a variety of estrogen responses and accompanying mechanisms related to human population. In the above report, the patient was a 28-year-old white man, fully masculinized, who had osteoporosis, unfused epiphyses and continuing linear growth in adulthood. He also had elevated serum estrogen, steroid and gonadotropin levels and no target-tissue responses to estrogen therapy. Genetic analysis showed that the patient was homozygous for a mutation in the second exon (R157X) of the ER gene, generating a premature stop codon. Similar to the observations in the ERKO mice, these findings suggested that estrogens in human males have a pivotal role in bone maturation and mineralization and are also important for complete epiphyseal closure. Therefore, normal androgen levels alone, as in this case, are not sufficient to promote skeletal maturation and retain bone mass, in contrast with the idea that androgens are important for the maintenance of bone mass in men. Data on the fertility of this patient were not available.

Activation of NK1.1+T Cellsin Vitroand Their Possible Role in Age-Associated Changes in Inducible IL-4 Production

Interleukin (IL)-12 or IL-4 produced early in an immune response directs the differentiation of naive antigen-activated CD4+T cells down a Th1 or Th2 pathway. The NK1.1+subset of T cells promptly produces IL-4 following activationin vivo.We demonstrate here that NK1.1+T cells can be directly induced to produce IL-4in vitrowhen activated under serum-free culture conditions. Platelet-derived growth factor in cell culture medium was inhibitory to the production of IL-4 by NK1.1+T cellsin vitro.Lymphocytes obtained from secondary lymphoid organs of aged mice produced greater quantities of IL-4 following stimulation than lymphocytes from mature adult animals. Aged mice expressed elevated percentages of NK1.1+T cells in their secondary lymphoid organs and peripheral blood. While this cell type was responsible for the total early IL-4 produced by lymphocytes from mature adult mice, both NK1.1+and memory phenotype (CD44high, CD45RBlow, NK1.1−) T cells from aged donors produced IL-4 following polyclonal T cell activation.

Methamphetamine Sensitization in Young Adult and Mature Adult Mice Assessed by Ambulation

The dose-effect relationship for the ambulation increase caused by methamphetamine (1–8 mg kg−1, s.c.), and the sensitization following the 10-time repeated administrations of methamphetamine (2 mg kg−1) at 3- to 4-day intervals were investigated in young adult (7-weeks old) and mature adult (7–9-months old) mice. The mature adult mice showed higher activity counts after the administration of methamphetamine (1 and 2 mg kg−1, but not 4 and 8 mg kg−1) than the young adult mice, although there were no marked differences in the time-courses of change in the increased ambulation. The repeated administration of 2 mg kg−1 methamphetamine induced sensitization to its ambulation-increasing effect in both the young adult and mature adult mice. However, the established sensitization was more marked in the young adult mice than in the mature adult mice. These results may be caused by a difference in the dopaminergic neurotransmission, or learning and memory ability between young adult and mature adult mice.