Effect of overdose zinc on mouse testis and its relation with sperm count and motility

The purpose of this study was to investigate the effects of excessive zinc intake on the testes and on sperm count and motility in mice. Thirty Balb c mice were divided randomly into 3 groups of 10 animals in each. Group I acted as controls; group II was supplied with drinking water containing 1.5 g/100 mL Zn, and group III was supplied with drinking water containing 2.5 g/100 mL Zn. The animals were sacrificed after 3 wk supplementation and the epididymis and testis were quickly excised. A negative correlation between Zn dose and sperm count and motility was found. The sperm count in group III was significantly lower than in groups II and I (p<0.05). The sperm motility in group III was significantly lower than in the controls (p<0.05). Degenerative changes, including spermatic arrest, degeneration of seminiferous tubules, and fibrosis in interstitial tissue, were observed in group III animals. These results show that high doses of zinc significantly alter sperm motility.     

电针对帕金森病模型小鼠黑质铁染色细胞和铁蛋白表达的影响

目的探讨电针防治帕金森病的作用机制。方法采用1-甲基-4-苯基1,2,3,6四氢吡啶腹腔注射建立帕金森病小鼠模型,利用组织化学技术以及免疫组织化学技术观察电针对帕金森病小鼠脑黑质铁染色细胞和铁蛋白表达的影响。结果模型组1周、2周、4周小鼠黑质铁染色阳性细胞的数量较正常组明显增多,染色强度也明显增强（P〈0．001;P〈0．01;P〈0．001）,电针在3个存活期内均可明显减少帕金森病小鼠黑质铁阳性细胞的数量和染色强度（P〈0．001;P〈0．05;P〈0．001）;模型组1周、2周黑质部位铁蛋白的表达与正常组相比都有不同程度下降（P〈0．01;P〈0．001）,而电针组2周帕金森病小鼠黑质铁蛋白的表达与模型组相比显著增加（P〈0．01）。结论电针可以通过降低脑内铁的含量,同时增加铁蛋白的表达,从而提高帕金森病模型小鼠脑抗氧化能力,达到保护黑质多巴胺能神经元的目的。     

Nonmuscle Myosin-Dependent Synthesis of Type I Collagen

Type I collagen, synthesized in all tissues as the heterotrimer of two α1(I) polypeptides and one α2(I) polypeptide, is the most abundant protein in the human body. Here we show that intact nonmuscle myosin filaments are required for the synthesis of heterotrimeric type I collagen. Conserved 5′ stem-loop in collagen α1(I) and α2(I) mRNAs binds the RNA-binding protein LARP6. LARP6 interacts with nonmuscle myosin through its C-terminal domain and associates collagen mRNAs with the filaments. Dissociation of nonmuscle myosin filaments results in secretion of collagen α1(I) homotrimer, diminished intracellular colocalization of collagen α1(I) and α2(I) polypeptides (required for folding of the heterotrimer), and their increased intracellular degradation. Inhibition of the motor function of myosin has similar collagen-specific effects, while disruption of actin filaments has a general effect on protein secretion. Nonmuscle myosin copurifies with polysomes, and there is a subset of polysomes involved in myosin-dependent translation of collagen mRNAs. These results indicate that association of collagen mRNAs with nonmuscle myosin filaments is necessary to coordinately synthesize collagen α1(I) and α2(I) polypeptides. We postulate that LARP6/myosin-dependent mechanism regulates the synthesis of heterotrimeric type I collagen by coordinating the translation of collagen mRNAs.     

A genomic reservoir for Tnfrsf genes is developmentally regulated and imprinted in the mouse

Tumor necrosis factor receptor superfamily is composed of at least 26 members in the mouse, three of which exist as a cluster within the imprinted Kcnq1 domain on chromosome 7. Tnfrsf22, 23 and 26 contain typical cystein-rich domains and Tnfrsf22 and 23 can bind ligands but have no signaling capacity. Thus, they are assumed to be decoy receptors. The developmental expression profile of these genes is unknown and knowledge of their imprinting patterns is incomplete and controversial. We found that all three genes are expressed during mouse embryonic development, and that they have a strong maternal bias, indicating that they may be affected by the KvDMR, the Kcnq1 imprinting control region. We found expression of an antisense non-coding RNA, {"type":"entrez-nucleotide","attrs":{"text":"AK155734","term_id":"74212930","term_text":"AK155734"}}AK155734, in embryos and some neonatal tissues. This RNA overlaps the Tnfrsf22 and possibly the Tnfrsf23 coding regions and is also expressed with a maternal bias. We were interested in exploring the evolutionary origins of the three Tnfrsf genes, because they are absent in the orthologous human Kcnq1 domain. To determine whether the genes were deleted from humans or acquired in the rodent lineage, we performed phylogenetic analyses. Our data suggest that TNFRSF sequences were duplicated and/or degenerated or eliminated from the KCNQ1 region several times during the evolution of mammals. In humans, multiple mutations (point mutations and/or deletions) have accumulated on the ancestral TNFRSF, leaving a single short non-functional sequence.     

Analysis of the mouse mutant Cloth-ears shows a role for the voltage-gated sodium channel Scn8a in peripheral neural hearing loss

Deafness is the most common sensory disorder in humans and the aetiology of genetic deafness is complex. Mouse mutants have been crucial in identifying genes involved in hearing. However, many deafness genes remain unidentified. Using N -ethyl N −nitrosourea (ENU) mutagenesis to generate new mouse models of deafness, we identified a novel semi-dominant mouse mutant, Cloth-ears ( Clth ). Cloth-ears mice show reduced acoustic startle response and mild hearing loss from ∼30 days old. Auditory-evoked brainstem response (ABR) and distortion product otoacoustic emission (DPOAE) analyses indicate that the peripheral neural auditory pathway is impaired in Cloth-ears mice, but that cochlear function is normal. In addition, both Clth/Clth and Clth/+ mice display paroxysmal tremor episodes with behavioural arrest. Clth/Clth mice also show a milder continuous tremor during movement and rest. Longitudinal phenotypic analysis showed that Clth/+ and Clth/Clth mice also have complex defects in behaviour, growth, neurological and motor function. Positional cloning of Cloth-ears identified a point mutation in the neuronal voltage-gated sodium channel α-subunit gene, Scn8a , causing an aspartic acid to valine (D981V) change six amino acids downstream of the sixth transmembrane segment of the second domain (D2S6). Complementation testing with a known Scn8a mouse mutant confirmed that this mutation is responsible for the Cloth-ears phenotype. Our findings suggest a novel role for Scn8a in peripheral neural hearing loss and paroxysmal motor dysfunction.     

Generation of cortactin floxed mice and cellular analysis of motility in fibroblasts

Cortactin is an F‐actin binding protein that has been suggested to play key roles in various cellular functions. Here, we generated mice carrying floxed alleles of the cortactin (Cttn) gene (Cttn^flox/flox mice). Expression of Cre recombinase in mouse embryonic fibroblasts (MEFs) isolated from Cttn^flox/flox embryos depleted cortactin within days, without disturbing F‐actin distribution and localization of multiple actin‐binding proteins. Cre‐mediated deletion of Cttn also did not affect cell migration. To obtain mice with a Cttn null allele, we next crossed Cttn^flox/flox mice with transgenic mice that express Cre recombinase ubiquitously. Western blot and immunocytochemical analysis confirmed complete elimination of cortactin expression in MEFs carrying homozygously Cttn null alleles. However, we found no marked alteration of F‐actin organization and cell migration in Cttn null‐MEFs. Thus, our results indicate that depletion of cortactin in MEFs does not profoundly influence actin‐dependent cell motility. genesis 47:638–646, 2009. © 2009 Wiley‐Liss, Inc.     

The Novel Use of a Heterozygous Knockout Mouse for Embryofetal Development Assessment of a Glucokinase Activator

Glucokinase activators (GKAs), such as AZD1656, are designed as antihyperglycemic agents for diabetics and can cause dose‐limiting hypoglycemia in normal animals used in embryofetal development studies. Genetically modified heterozygous GK knockout (gk^del/wt) mice are less susceptible to severe GKA‐induced hypoglycemia than wild‐type mice due to their elevated baseline glucose levels. In this study, the gk^del/wt mouse was used as an alternative rodent strain for embryofetal development studies with AZD1656. Heterozygous global knockout gk^del/wt females were dosed with 20, 50, or 130 mg/kg/day of AZD1656 or vehicle for a minimum of 14 consecutive days before mating with wild‐type males and throughout organogenesis. Maternal effects were confined to slightly reduced food consumption, reduced body weight gain, and the pharmacologic effect of decreased plasma glucose. Fetuses were genotyped. Fetal weights at the high dose were slightly reduced but there was no effect on fetal survival. There were two specific major malformations, omphalocele and right‐sided aortic arch, with increased fetal incidence in mid‐ and high‐dose fetuses (e.g., omphalocele fetal incidence of 0.6, 0.7, 4.6, and 2% across the dose groups) plus increased incidences of minor abnormalities and variants indicative of either delayed or disturbed development. Fetal weight and abnormalities were unaffected by fetal genotype. The fetal effects are considered hypoglycemia related. There was no effect on embryofetal survival in the gk^del/wt mouse at AZD1656 exposures, which were 70× higher than those causing 75% fetal death in rabbits. This illustrates the value of genetically modified animals in unraveling target versus chemistry‐related effects.     

Deletion of peptide amidation enzymatic activity leads to edema and embryonic lethality in the mouse

Peptidylglycine alpha-amidating monooxygenase (PAM) catalyzes the COOH-terminal amidation of peptide hormones. We previously had found high expression of PAM in several regions of the developing rodent. To determine the function of PAM during mouse embryogenesis, we produced a null mutant of the PAM gene. Homozygous mutants die in utero between e14.5 and e15.5 with severe edema that is likely due to cardiovascular deficits. These defects include thinning of the aorta and carotid arteries and are very similar to those of the recently characterized adrenomedullin (AM) gene KO despite the presence of elevated immunoreactive AM in PAM KO embryos. No peptide amidation activity was detected in PAM mutant embryos, and there was no moderation of the AM-like phenotype that could be expected if any alternative peptide amidation mechanism exists in the mouse. Despite the proposed contribution of amidated peptides to neuronal cell proliferation, no alteration in neuroblast proliferation was observed in homozygous mutant embryos prior to lethality. Mice heterozygous for the mutant PAM allele develop normally and express wildtype levels of several amidated peptides despite having one half the wildtype levels of PAM activity and PAM protein. Nonetheless, both an increase in adiposity and a mild glucose intolerance developed in aged (>10 months) heterozygous mice compared to littermate controls. Ablation of PAM thus demonstrates an essential function for this gene during mouse development, while alterations in PAM activity in the adult may underlie more subtle physiologic effects.     

Knockdown of RBBP7 unveils a requirement of histone deacetylation for CPC function in mouse oocytes

During mouse oocyte maturation histones are deacetylated, and inhibiting this deacetylation leads to abnormal chromosome segregation and aneuploidy. RBBP7 is a component of several different complexes that contain histone deacetylases, and therefore could be implicated in histone deacetylation. We find that Rbbp7 is a dormant maternal mRNA that is recruited for translation during oocyte maturation to regulate the histone deacetylation. Importantly, we show that the maturation-associated decrease of histone acetylation is required for localization and function of the chromosomal passenger complex (CPC) during oocyte meiotic maturation. This finding can explain the phenotypes of oocytes where Rbbp7 is depleted by an siRNA/morpholino cocktail including severe chromosome misalignment, improper kinetochore–microtubule attachments, impaired SAC function, cytokinesis defects, and increased incidence of aneuploidy at metaphase II (Met II). These results implicate RBBP7 as a novel regulator of histone deacetylation during oocyte maturation and provide evidence that such deacetylation is required for proper chromosome segregation by regulating localized CPC function.