Male WT Mice Research Articles

Progressive Triad-Mitochondria Un-Coupling in Aging

An impairment of the mechanisms controlling the release of calcium from internal stores (excitation-contraction (EC) coupling) has been proposed to contribute to the age-related decline of muscle performance that accompanies aging (EC un-coupling theory). EC coupling in muscle fibers occurs at specialized intracellular junctions called calcium release units, or triads, which are specifically placed at sarcomere's I-A band transition. In recent publications we have shown that: a) in human muscle, the frequency of triads decreases significantly with age (Boncompagni et al., 2006; J Gerontol 61:995); and b) in mice, triads are tethered to mitochondria placed at the I band (Boncompagni et al., 2009; MBC 20:1059). Here we have studied the frequency, sarcomeric-localization, ultrastructure, and coupling of triads/mitochondria in EDL from male WT mice using transmission electron microscopy (TEM).Preliminary results indicates that the number of triads/100μm2 of longitudinal section in aging mice (n=4, 25-35 months of age) decreases compared to the adult mice (n=5, 3-12 months of age): 92±9 vs. 79±8. In addition, the percentage of abnormally positioned triads (longitudinal and/or oblique) increases. On the other hand, the total volume of mitochondria does not change significantly with age. However, the number of mitochondria-profiles/100μm2 of longitudinal section is reduced (54±7 vs. 43±6), suggesting a remodelling/fusion of these organelles. Finally, we have assessed the positioning of mitochondria in respect to myofibrils and triads: a) the number of mitochondria at the A band (misplaced) slightly increases with age (9% vs 3%), whereas the number of triads-mitochondria couples is significantly reduced: 39±5 vs. 26±4. Our observations indicates: a) a age-related partial disarrangement and spatial re-organization of EC coupling/mitochondrial apparatuses; and b) a decreased percentage of mitochondria functionally tethered to calcium release sites. This could in part explain the decline of muscle performance associated to increasing age.

Gene expression profiling of dilated cardiomyopathy in older male EP4 knockout mice

Using a line of mice with cardiac-specific knockout (KO) of the EP4 receptor gene, experiments were designed to determine whether a cardiac phenotype developed with age. Cardiac function was assessed by echocardiography in 23- to 33-wk-old male and female KO and littermate controls (WT) mice. After echocardiography, hearts were removed to assess weight, and then some were further processed for histology [myocyte cross-sectional area (MCSA), interstitial collagen fraction (ICF), and macrophage infiltration] and some for extraction of total RNA and protein. Older male KO mice had reduced ejection fraction (EF) coupled with left ventricular dilatation. MCSA and infiltrating macrophages were not different between groups, but ICF increased by 39% in KO mice. In contrast to male KO mice, 30- to 32-wk-old female KO mice had only a slight reduction in EF. To understand gene expression differences between male WT and KO mice, we performed whole genome gene expression profiling (Illumina BeadChips) on hearts of 30-to 32-wk-old mice. Data indicated that 156 genes were overexpressed in the KO hearts more than twofold, including genes involved in remodeling, inflammation, and oxidative stress. Overexpressed chemokines/cytokines were further examined in hearts of 10- to 12-wk-old male KO mice, and we found that growth differentiation factor-15 (GDF-15) expression was higher in KO than in WT hearts. In conclusion, EP4 knockdown in cardiac myocytes in aged male KO mice is in part associated with increased fibrosis, reduced EF, and dilated cardiomyopathy. Early overexpression of GDF-15 in hearts of male KO mice may contribute to or be a marker of the disease phenotype. The absence of serious cardiac dysfunction in aged female mice suggests a sexual dimorphism in the phenotype.

Male mice with deleted Wolframin (Wfs1) gene have reduced fertility

BackgroundWolfram Syndrome (WS) is an autosomal recessive disorder characterised by non-autoimmune diabetes mellitus, optic atrophy, cranial diabetes insipidus and sensorineural deafness. Some reports have described hypogonadism in male WS patients. The aim of our study was to find out whether Wfs1 deficient (Wfs1KO) male mice have reduced fertility and, if so, to examine possible causes.MethodsWfs1KO mice were generated by homologous recombination. Both Wfs1KO and wild type (wt) male mice were mated with wt female mice. The number of litters and the number of pups were counted and pregnancy rates calculated. The motility and morphology of the sperm and the histology of testes were analysed. Serum testosterone and FSH concentrations were also measured.ResultsThe pregnancy rate in wt females mated with Wfs1KO males was significantly lower than in the control group (15% vs. 32%; p < 0.05), but there was no significant difference in litter size. Analysis of male fertility showed that, in the Wfs1KO group, eight males out of 13 had pups whereas in the control group all 13 males had at least one litter. Sperm motility was not affected in Wfs1KO mice, but Wfs1KO males had less proximal bent tails (14.4 +/- 1.2% vs. 21.5 +/- 1.3 p < 0.05) and less abnormal sperm heads (22.8 +/- 1.8 vs. 31.5 +/- 3.5, p < 0.05) than wt males. Testes histology revealed significantly reduced number of spermatogonia (23.9 +/- 4.9 vs. 38.1 +/- 2.8; p < 0.05) and Sertoli cells (6.4 +/- 0.5 vs. 9.2 +/- 1.0; p < 0.05) in Wfs1KO mice. Serum testosterone and FSH concentrations did not differ between the two groups.ConclusionThe impaired fertility of Wfs1KO male mice is most likely due to changes in sperm morphology and reduced number of spermatogenic cells. The exact mechanism through which the Wfs1 gene influences sperm morphology needs to be clarified in further studies.

Epididymis seleno-independent glutathione peroxidase 5 maintains sperm DNA integrity in mice

The mammalian epididymis provides sperm with an environment that promotes their maturation and protects them from external stresses. For example, it harbors an array of antioxidants, including non-conventional glutathione peroxidase 5 (GPX5), to protect them from oxidative stress. To explore the role of GPX5 in the epididymis, we generated mice that lack epididymal expression of the enzyme. Histological analyses of Gpx5-/- epididymides and sperm cells revealed no obvious defects. Furthermore, there were no apparent differences in the fertilization rate of sexually mature Gpx5-/- male mice compared with WT male mice. However, a higher incidence of miscarriages and developmental defects were observed when WT female mice were mated with Gpx5-deficient males over 1 year old compared with WT males of the same age. Flow cytometric analysis of spermatozoa recovered from Gpx5-null and WT male mice revealed that sperm DNA compaction was substantially lower in the cauda epididymides of Gpx5-null animals and that they suffered from DNA oxidative attacks. Real-time PCR analysis of enzymatic scavengers expressed in the mouse epididymis indicated that the cauda epididymidis epithelium of Gpx5-null male mice mounted an antioxidant response to cope with an excess of ROS. These observations suggest that GPX5 is a potent antioxidant scavenger in the luminal compartment of the mouse cauda epididymidis that protects spermatozoa from oxidative injuries that could compromise their integrity and, consequently, embryo viability.

Epitope-tagged Receptor Knock-in Mice Reveal That Differential Desensitization of α2-Adrenergic Responses Is because of Ligand-selective Internalization

Although ligand-selective regulation of G protein-coupled receptor-mediated signaling and trafficking are well documented, little is known about whether ligand-selective effects occur on endogenous receptors or whether such effects modify the signaling response in physiologically relevant cells. Using a gene targeting approach, we generated a knock-in mouse line, in which N-terminal hemagglutinin epitope-tagged alpha(2A)-adrenergic receptor (AR) expression was driven by the endogenous mouse alpha(2A)AR gene locus. Exploiting this mouse line, we evaluated alpha(2A)AR trafficking and alpha(2A)AR-mediated inhibition of Ca(2+) currents in native sympathetic neurons in response to clonidine and guanfacine, two drugs used for treatment of hypertension, attention deficit and hyperactivity disorder, and enhancement of analgesia through actions on the alpha(2A)AR subtype. We discovered a more rapid desensitization of Ca(2+) current suppression by clonidine than guanfacine, which paralleled a more marked receptor phosphorylation and endocytosis of alpha(2A)AR evoked by clonidine than by guanfacine. Clonidine-induced alpha(2A)AR desensitization, but not receptor phosphorylation, was attenuated by blockade of endocytosis with concanavalin A, indicating a critical role for internalization of alpha(2A)AR in desensitization to this ligand. Our data on endogenous receptor-mediated signaling and trafficking in native cells reveal not only differential regulation of G protein-coupled receptor endocytosis by different ligands, but also a differential contribution of receptor endocytosis to signaling desensitization. Taken together, our data suggest that these HA-alpha(2A)AR knock-in mice will serve as an important model in developing ligands to favor endocytosis or nonendocytosis of receptors, depending on the target cell and pathophysiology being addressed.

The effect of annexin 1 on inflammatory stress in the microcirculation

The bodies' response mechanisms against inflammation can be influenced by gender and sex hormone status, and can become deregulated and detrimental, leading to tissue damage. This study has investigated the anti‐inflammatory protein annexin 1 and its role in the sexual dimorphism of inflammatory disease.Male and proestrous female WT (C57BL/6) and male AnxA1 null (ANXA1‐/‐) mice were challenged with lipopolysaccharide (LPS). The mesentery was exteriorised and leukocyte‐endothelial cell interactions were recorded using intravital microscopy.In both male and female WT mice, LPS caused a significant increase in leukocyte adherence and emigration in venules vs. saline group, but with no difference between sexes. LPS administration had a modest effect in AnxA1‐/‐ mice, with only venular leukocyte rolling being decreased, which may be indicative of the initial phase of inflammation. Further studies in which the time course of the response to LPS is examined are now required. However, our findings fit with previous data concerning the survival rate of AnxA1‐/‐ mice, which is significantly decreased at 24hr, but not 2hr (1).To conclude, these data suggest that there is no sexual dimorphism in the inflammatory response following 2hr LPS challenge. Furthermore, this inflammatory response is not observed in AnxA1‐/‐ mice.1. Damazo AS et al., Am J Pathol. 2005;166:1607‐17.

G020 Properties of pulmonary artery smooth muscle from task-1 knockout mice

In pulmonary artery (PA), compelling evidence have suggested that TWIK-related Acid Sensitive K+ -1 (TASK-1) channel contribute to the background K+ current that support the membrane potential (Em) of the smooth muscle cells. However, due to poor selectivity of its modulators, the role of TASK-1 channel in the functional regulation of tone in pulmonary arteries remains elusive. The purpose of this study was to investigate the properties of PA isolated from mice in which the TASK-1 and TASK-3 genes have been deleted (TASK1/3 knockout, KO), compared with their wild type controls (C57BL/6 mice, WT). Intra-PAs were isolated from adult male WT and KO mice. Vessels were mounted on a wire myograph for isometric tension recordings. PAs were also processed for gene expression studies using RT-PCR. The addition of 10mM KCl, the K+ channel modulators 4-AP (1mM) and levcromakalim (10μm), or the L-type calcium channel blocker nifedipine (1μm), did not elicit any significant contractile response in PAs from either KO (N=3) or WT mice (N=3), suggesting that the Em is not depolarised and that artery is fully dilated in both groups. The contractile response of PA to phenylephrine, serotonin and PGF2α did not show any difference between the two strains. Potency (pEC50) of the prostacyclin analog treprostinil to induce relaxation on preconstricted vessels was significantly higher in KO mice (6.7 ± 0.1, N = 3) than in WT mice (7.2 ± 0.1N = 4). RT-PCR confirmed TASK-1 expression in PA from WT but not KO mice whereas TASK-3 was not expressed in either WT or KO mouse PA. TASK-2, TASK-5, TWIK-2, Kv1.5 and Kv2.1 RNA levels did not show any significant alteration between WT (N = 3) or KO mice (N = 3). The PA isolated from KO mice does not display any resting tone or altered responses to the studied agonists. Surprisingly, response to treprostinil was increased, and TASK-1 absence in PA was not compensated by alteration of other K+ channels expression. The results thus far do not support a major physiological role for TASK-1 in mouse PA.

Deletion of the Maternally Expressed Imprinted Gene, Gtl2, Resulted in Parental-specific Lethality in Mice.

In mammals, parental-specific methylation imprinting, which occurs independently during male and female gametogenesis, regulates monoallelic expression of imprinted genes. It is well known that many imprinted genes are involved in embryonic development. In mice, the Dlk1-Gtl2 domain spanning 1 Mb and located in the distal region of chromosome 12 contains a cluster of imprinted genes: the paternally expressed genes Dlk1, Rtl1, and Dio3 and the maternally expressed genes Gtl2, antiRtl1, Rian, and Mirg. The observations that maternal and paternal duplications in this region resulted in perinatal origin-dependent lethality, indicate that the Dlk1-Gtl2 domain plays an essential role in peri- and postnatal development. Furthermore, studies using gene knockout mice have shown that Dlk1 is responsible for growth and skeletal formation, and that Dio3 contributes to the metabolism of thyroid hormones. However, little is known regarding the function of the maternally expressed imprinted genes Gtl2, antiRtl1, Rian, and Mirg, except that they are non-coding RNA. Here, we generated mice harboring a deletion that spanned exons 1-5 by using a neomycin-resistance cassette and carried out mating tests in order to elucidate the function of Gtl2. Mice inheriting the mutation from either the father (Gtl2 Pat-KO) or the mother (Gtl2 Mat-KO) were produced by mating WT female mice × heterozygous mutant male mice (n = 12) and heterozygous female mice × WT male mice (n = 4), respectively. In the Gtl2 Pat-KO mice, the mean number of offspring per litter was significantly lower (2.7/litter, n = 12) than that of WT mice (4.9/litter, n = 12), suggesting loss of the embryos before birth. Furthermore, the pups showed severe pre- and postnatal growth retardation, and half the pups died within 1 day of birth. However, the survivors grew up to be fertile adults. On the other hand, in the Gtl2 Mat-KO mice, the pups were born with normal phenotypes, showing normal litter size and body weight. However, they died within 4 weeks of birth and showed severe growth retardation at this time. In order to understand these parental-origin-specific phenotypes, we examined the expression of the imprinted genes in the 1-day-old pups by using quantitative RT-PCR. In the Gtl2 Pat-KO mice, the expression of Dlk1 and Dio3, which are involved in growth, was reduced to half the levels observed in the controls. In the Gtl2 Mat-KO mice, in addition to the lack of Gtl2 expression, the expression levels of Rian and Mirg were significantly reduced. It is known that these 2 maternal transcripts contain a number of snoRNAs and miRNAs, although the function of these remains unknown. The present data, however, suggested that non-coding RNA, including Gtl2, is involved in postnatal development. The present results implicate Gtl2, for the first time, in the regulation of gene expression of the Dlk1-Gtl2 domain and causation of parental-origin-specific fetal and postnatal lethality in mice.

CORRIGENDUM

Liver InternationalVolume 28, Issue 4 p. 582-582 Free Access CORRIGENDUM This article corrects the following: Overexpression of NK2 promotes liver fibrosis in carbon tetrachloride-induced chronic liver injury Satoshi Hagiwara, Toshiyuki Otsuka, Yuichi Yamazaki, Takashi Kosone, Naondo Sohara, Takeshi Ichikawa, Ken Sato, Satoru Kakizaki, Hitoshi Takagi, Masatomo Mori, Volume 28Issue 1Liver International pages: 126-131 First Published online: November 1, 2007 First published: 12 March 2008 https://doi.org/10.1111/j.1478-3231.2008.01689.xAboutSectionsPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinkedInRedditWechat In Hagiwara et al. (1), the following errors were published: Material and methods Animals In the fibrosis model, 6-week-old male NK2 transgenic mice (n=3) and male WT mice (n=3) received intraperitoneal injection of 1 ml/kg CCl4 (Kanto Chemistry, Tokyo, Japan) dissolved in olive oil (Kanto Chemistry) twice a week. The text was incorrect and should have read: In the fibrosis model, 6-week-old male NK2 transgenic mice (n=4–6) and male WT mice (n=3–5) received intraperitoneal injection of 1 ml/kg CCl4 (Kanto Chemistry, Tokyo, Japan) dissolved in olive oil (Kanto Chemistry) twice a week. Statistical analysis All experimental data are expressed as mean±SD. Differences between groups were examined for statistical significance using Student's t-test. The text was incorrect and should have read: All experimental data are expressed as mean±SD. Differences between groups were examined for statistical significance using Student's t-test or analysis of variance (anova) followed by Scheffe's test. Results Figure 2 was presented incorrectly (see changes above). Figure 2 should have been presented as follows: Figure 1Open in figure viewerPowerPoint The authors apologise for these errors. Reference 1 Hagiwara S, Otsuka T, Yamazaki Y, et al. Overexpression of NK2 promotes liver fibrosis in carbon tetrachloride-induced chronic liver injury. Liver Int 2008; 28: 126– 31. Volume28, Issue4April 2008Pages 582-582 FiguresReferencesRelatedInformation

Role of transcription factors in estrogen‐dependent upregulation of CYP 2C family in arteries of eNOS deficient mice

Upregulation of cytochrome P450 (CYP) 2C29/38/40 is responsible for the EET‐mediated flow‐dependent dilation in microvessels of female mice, as a function of NO deficiency. In the present studies, we provide evidence of the correlation between transcription factors (PPAR‐γ, PPAR‐δ and RXR‐γ) and CYP 2C29/38/40 in isolated arterioles of female eNOS‐KO mice by microarray (SuperArray, OMM‐401). Five single arterioles isolated from mesentery of both genders of eNOS‐KO and WT mice were pooled and snap frozen in liquid nitrogen; ∼10 μg of total RNA were isolated, and a total of 10 arrays from 2 WT and 3 eNOS‐KO mice of each gender were performed. Microarray results indicate that associated with a specific upregulation of CYP 2C29/38/40, expression of genes for PPAR‐γ, PPAR‐δ and RXR‐γ, the nuclear receptors that function as gene regulators via specific DNA binding, was also increased (P&lt;0.01, 0.05 and 0.03, respectively) in female eNOS‐KO compared to male eNOS‐KO vessels. These significant differences were validated by quantitative real‐time PCR and Western blot analysis. The increase in gene expression for the three transcription factors was only observed in vessels of female, but not in those of male eNOS‐KO mice, and without significant differences between vessels of WT male and WT female mice, a pattern that is similar to the upregulation of CYP 2C/29/38/40. Thus, the microarray analysis screened the global expression of genes for the transcription factors and revealed the specificity of estrogen and NO deficiency in the control of the transcription factors which may play a role in the regulation of CYP 2C expression (Supported by NIH HL‐43023, HL‐68813 and HL‐070653).

The skeletal response to estrogen is impaired in female but not in male steroid receptor coactivator (SRC)-1 knock out mice

Estrogen (E) is critical for the maintenance of bone mass in both female and male mice and steroid receptor coactivator (SRC)-1 has been shown to be important for mediating E effects on bone, at least in female mice. In the present study, we defined the skeletal phenotype of male SRC-1 knock out (KO) mice and compared it with their female littermates. Further, to determine the role of SRC-1 in mediating effects of E on bone in male mice, we examined the skeletal effects of gonadectomy (gnx) with or without E replacement in male mice and placed these findings in the context of our previous studies in female SRC-1 KO mice. Analysis of a large group of male (WT, n = 67; SRC-1 KO, n = 56) and female (WT, n = 66; SRC-1 KO, n = 70) mice showed a significant decrease in trabecular volumetric bone mineral density (vBMD) in SRC-1 KO mice compared to their WT littermates in both genders (male SRC-1 KO, 275 ± 3 vs. WT, 295 ± 3 mg/cm 3, P < 0.001; female SRC-1 KO, 210 ± 2 vs. WT, 221 ± 2 mg/cm 3, P < 0.001). Following gnx and E replacement (10 μg/kg/day), we previously demonstrated that SRC-1 KO female mice have a defect in E action in trabecular, but not in cortical bone. In contrast, we now demonstrate that the same dose of E administered to gnx'd male SRC-1 KO mice was sufficient to prevent trabecular bone loss in these mice. For example, in WT female mice, gnx followed by E replacement maintained spine BMD (1.2 ± 3.4% vs. baseline) as compared to gnx without E replacement (− 12.7 ± 2.6%, P < 0.001 vs. sham); this effect of E was absent in SRC-1 KO female mice. By contrast, the identical dose of E was equally effective in maintaining spine BMD in E-treated gnx'd male WT (− 5.2 ± 5.1% vs. baseline) and male SRC-1 KO (− 5.4 ± 5.3%) mice, respectively, as compared to gnx'd mice without E treatment (WT, − 17.6 ± 2.5%, P = 0.02; SRC-1 KO, − 28.6 ± 2.6%, P < 0.001 vs. sham). E treatment was effective in suppressing cancellous bone turnover in both gnx'd WT and SRC-1 KO male mice as determined by significant reductions in osteoblast and osteoclast numbers; however, in female mice, E treatment only suppressed bone turnover in WT but not in SRC-1 KO mice. Collectively, these findings demonstrate that loss of SRC-1 results in trabecular osteopenia in male and female mice, but in contrast to female mice, this is not due to any detectable resistance to E action in trabecular bone in male SRC-1 KO mice.

Protease-Activated Receptor-2 Augments Experimental Crescentic Glomerulonephritis

Protease-activated receptor-2 (PAR-2) is a cellular receptor expressed prominently on epithelial, mesangial, and endothelial cells in the kidney and on macrophages. PAR-2 is activated by serine proteases such as trypsin, tryptase, and coagulation factors VIIa and Xa. It induces pleiotropic effects including vasodilatation, increasing plasminogen activator inhibitor (PAI-1) expression, mesangial cell proliferation, and cytokine production by macrophages. The role of PAR-2 in renal inflammation was studied in antiglomerular basement membrane antibody-induced crescentic glomerulonephritis (CGN) using PAR-2-deficient (PAR-2(-/-)) mice and wild-type littermate controls. PAR-2(-/-) mice had reduced crescent formation, proteinuria, and serum creatinine compared with wild-type mice 21 days after initiation of CGN. Glomerular accumulation of CD4(+) T cells and macrophages and the number of proliferating cells in glomeruli were similar in both groups. Glomerular fibrin deposition was significantly reduced in PAR-2(-/-) mice, and this was associated with reduced renal plasminogen activator inhibitor expression and increased renal matrix-metalloprotinase-9 activity. These results demonstrate a proinflammatory role for PAR-2 in CGN that is independent of effects on glomerular leukocyte recruitment and mesangial cell proliferation. PAR-2-mediated augmentation of renal plasminogen activator inhibitor expression and inhibition of matrix-metalloprotinase-9 activity may contribute to increased glomerular fibrin accumulation and glomerular injury in CGN.

A previously uncharacterized role for estrogen receptor β: Defeminization of male brain and behavior

Sex differences in brain and behavior are ubiquitous in sexually reproducing species. One cause of sexual dimorphisms is developmental differences in circulating concentrations of gonadal steroids. Neonatal testes produce androgens; thus, males are exposed to both testosterone and estradiol, whereas females are not exposed to high concentrations of either hormone until puberty. Classically, the development of neural sex differences is initiated by estradiol, which activates two processes in male neonates; masculinization, the development of male-type behaviors, and defeminization, the loss of the ability to display female-type behaviors. Here, we test the hypothesis that defeminization is regulated by estrogen receptor beta (ERbeta). Adult male ERbeta knockout and WT mice were gonadectomized, treated with female priming hormones, and tested for receptive behavior. Indicative of incomplete defeminization, male ERbeta knockout mice showed significantly higher levels of female receptivity as compared with WT littermates. Testes-intact males did not differ in any aspects of their male sexual behavior, regardless of genotype. In olfactory preference tests, males of both genotypes showed equivalent preferences for female-soiled bedding. Based on these results, we hypothesize that ERbeta is involved in defeminization of brain and behavior. This aspect of ERbeta function may lead to developments in our understanding of neural-based sexually dimorphic human behaviors.

Dopamine D5 receptor modulates male and female sexual behavior in mice

Dopamine exerts its actions through at least five receptor (DAR) isoforms. In female rats, D5 DAR may be involved in expression of sexual behavior. We used a D5 knockout (D5KO) mouse to assess the role of D5 DAR in mouse sexual behavior. Both sexes of D5KO mice are fertile and exhibit only minor disruptions in exploratory locomotion, startle, and prepulse inhibition responses. This study was conducted to characterize the sexual behavior of male and female D5KO mice relative to their WT littermates. Female WT and D5KO littermates were ovariectomized and given a series of sexual behavior tests after treatment with estradiol benzoate (EB) and progesterone (P). Once sexual performance was optimal the dopamine agonist, apomorphine (APO), was substituted for P. Male mice were observed in pair- and trio- sexual behavior tests. To assess whether the D5 DAR is involved in rewarding aspects of sexual behavior, WT and D5KO male mice were tested for conditioned place preference. Both WT and D5KO females can display receptivity after treatment with EB and P, but APO was only able to facilitate receptivity in EB-primed WT, not in D5KO, mice. Male D5KO mice display normal masculine sexual behavior in mating tests. In conditioned preference tests, WT males formed a conditioned preference for context associated with either intromissions alone or ejaculation as the unconditioned stimulus. In contrast, D5KO males only showed a place preference when ejaculation was paired with the context. In females, the D5 DAR is essential for the actions of dopamine on receptivity. In males, D5 DAR influences rewarding aspects of intromissions. Taken together, the work suggests that the D5 receptor mediates dopamine's action on sexual behavior in both sexes, perhaps via a reward pathway.

CYSTOMETRIC FINDINGS IN MICE LACKING MUSCARINIC M 2 OR M 3 RECEPTORS

The physiological importance of muscarinic M3 and M2 receptors for bladder function was investigated in vivo using mice lacking M3 or M2 receptors and littermate WT controls. Unanesthetized mice of each sex underwent continuous cystometry before and after administration of atropine (1 mg/kg). Male M3 knockout (KO) mice had longer voiding intervals, and larger micturition volumes and bladder capacity than M2 KO or WT males. There was no significant difference in any cystometric parameters between male M2 KO and WT mice. In females M3 KO and M2 KO mice had longer voiding intervals and larger micturition volumes than WT animals. Atropine had marked inhibitory effects on voiding efficacy in WT and M2 KO mice but it had no effect on any cystometric parameters in M3 KO mice. The current results confirm that M3 receptor is the principal muscarinic receptor subtype responsible for bladder contraction and the role of M2 receptors is of minor importance. Functional impairments found in M3 KO mice were milder than those elicited by acute blockade of muscarinic receptors by atropine in WT mice, suggesting that noncholinergic mechanisms can compensate for a chronic loss of M3 receptors.

Superoxide generation from Kupffer cells contributes to hepatocarcinogenesis: studies on NADPH oxidase knockout mice

We hypothesized that superoxide from Kupffer cells (KC) contributes to hepatocarcinogenesis. p47phox(-/-) mice, deficient in phagocyte NADPH oxidase and superoxide generation, received a single dose of the hepatocarcinogen diethylnitrosamine (DEN). The following hepatic effects were observed at time points between 30 min and 35 days. Liver damage after DEN was manifested by loss of body and liver mass and of liver DNA and by an increase in apoptosis, necrosis and signs of inflammation. These effects were massive in wild-type (wt) male mice, but only very mild in p47phox(-/-) mice. Regenerative DNA synthesis subsequent to liver damage was high in wt male mice, but weak in p47phox(-/-) mice. In females the apparent protection by p47phox(-/-) was less pronounced than in males. Therefore, further experiments were performed with males. In KC isolated from wt mice superoxide production was enhanced by DEN pretreatment in vivo. Also, in vitro addition of DEN to KC cultures induced superoxide release, similarly to lipopolysaccharide, a standard KC activator. Thus, DEN directly activates wt KC to produce superoxide. KC from p47phox(-/-) mice did not release superoxide. TNFalpha production by isolated KC was transiently depressed 0.5 h after DEN treatment in vivo, but recovered rapidly. In blood serum TNFalpha levels of wt mice were elevated for the initial 6 h. TNFalpha in KC cultures and in serum of p47phox(-/-) mice was reduced. DEN in vivo induced DNA damage ('comets') in hepatocytes. This damage was extensive in wt mice but much less in p47phox(-/-) mice. These studies suggest two conclusions: (i) superoxide generation by phagocytes during liver damage and inflammation aggravates genotoxic and cytotoxic effects in hepatocytes and may thus contribute to tumor initiation and promotion; (ii) DEN has a direct stimulatory effect on KC to release superoxide and TNFalpha.

Estrogen receptor-alpha, sexual dimorphism and reduced-size liver ischemia and reperfusion injury in mice.

Estrogen (E(2)) exerts its effect on target organs principally by interacting with specific estrogen receptors (ER) such as ER-alpha or ER-beta. The role that these E(2) receptors play in mediating the protective effects observed in RSL+I/R induced injury remains to be defined. To study the role of ER-alpha, we anesthetized female and male wild type (wt; C57Bl/6) and ER-alpha-deficient (alphaERKO) mice and subjected them to 70% liver ischemia for 45 min followed by resection of the remaining 30% nonischemic lobes and reperfusion of the ischemic tissue. For some experiments, wt and alphaERKO male mice were injected with E(2). Survival was monitored on a daily basis while liver injury was assessed by quantifying serum alanine aminotransferase (ALT) levels and histopathology. Hepatic eNOS mRNA levels were evaluated using semi-quantitative RT-PCR. Our data showed that untreated females or males treated with E(2) survived RSL+I/R surgery indefinitely whereas all male mice given vehicle died within 3-5 days following surgery. This protective effect was diminished in alphaERKO female mice such that only 40% of alphaERKO females survived 7 d following RSL+I/R. Furthermore, liver injury was significantly higher in alphaERKO females compared with their wt counterparts and similar to those seen in wild type males and alphaERKO males. The protective effect observed in wild type females or E(2) treated males correlated well with increases in hepatic eNOS message whereas both male and female alphaERKO mice exhibited significantly lower levels of eNOS mRNA. We conclude that this protection may in part be due to the E(2)/ER-alpha-mediated activation of eNOS.

Early Orchiopexy Restores Fertility in the Hoxa 11 Gene Knockout Mouse

We investigated whether infertility could be reversed in cryptorchid mice (with disrupted expression of the homeobox gene Hoxa 11) by orchiopexy and mating such animals with females of proven fertility. Hoxa 11 mutant and WT male mice were genotyped by polymerase chain reaction. Surgery (orchiopexy or sham operation) was performed at age 18 days and fertility was assessed at ages 6 to 8 weeks. Animals were sacrificed at ages 6 to 9 months and computer assisted semen analysis was performed on fluid obtained by epididymal puncture. Five of 28 mutant mice proved fertile following orchiopexy versus 0 of 22 after sham operation (p <0.05). Values in WT mice were 18 of 35 and 25 of 33, respectively (p <0.01). Mean spermatozoa counts +/- SEM were 21.7 +/- 5.9 x 106/ml in 8 mutant mice with orchiopexy, 2.78 +/- 1.59 x 106/ml in 8 sham operated mutant mice (p <0.002), 15.6 +/- 4.9 x 106/ml in 7 WT mice with orchiopexy and 36.3 +/- 10.5 x 106/ml in 9 sham operated WT mice (p <0.02). Testicular position following orchiopexy is important to achieve fertility but the surgical procedure was associated with a degree of damage. Since mutant animals did not attain the fertility rates observed in WT animals following orchiopexy, other factors (possibly vaso-epididymal) may be necessary for normal spermatogenesis. Further studies of this model may allow the identification of such factors.

Conference Proceedings Available

Sex steroid hormones secreted by gonads influence development and expression of many behaviors including parental behaviors. The capacity to display many behaviors develops under the influence of sex steroid hormones; it begins with gonadal differentiation and lasts through puberty. The timing of gonadectomy may have important and long lasting effects on the organization and activation of neural circuits regulating the expression of different behaviors. The present study investigated the importance of exposure to endogenous gonadal steroid hormones during pubertal period/adolescence on parental behavior in adult mice. Male and female WT mice were gonadectomized either before puberty (25 days of age) or after puberty (60 days of age) and tested for parental behavior with and without estradiol benzoate (EB) replacement in adulthood. Additional groups of mice were gonadectomized at P25 and supplemented with estradiol (females) or testosterone (males) during puberty. Female mice gonadectomized after puberty or gonadectomized before puberty and supplemented with estradiol during puberty, displayed better pup directed parental behaviors in comparison to mice gonadectomized at 25 days of age regardless of treatment with estradiol in adulthood. However, mice treated with EB in adulthood displayed better non-pup directed nest building behavior than when they were tested without EB treatment regardless of sex and time of gonadectomy. To examine whether the sensitivity to sex steroid hormones was altered due to differences in time without gonads prior to the testing, mice were also tested for female sex behavior and there were no differences between mice gonadectomized at P25 or P60, although this could not completely rule out the possibility that parental behavior is more sensitive to prolonged absence of steroid hormones than female sex behavior. These results suggest that the absence of gonads and thereby the absence of appropriate gonadal steroid hormones during puberty/adolescence may have a profound effect on pup directed parental behaviors in adult mice.