Deletion of Genes Implicated in Protecting the Integrity of Male Germ Cells Has Differential Effects on the Incidence of DNA Breaks and Germ Cell Loss

Infertility affects approximately 20% of couples in Europe and in 50% of cases the problem lies with the male partner. The impact of damaged DNA originating in the male germ line on infertility is poorly understood but may increase miscarriage. Mouse models allow us to investigate how deficiencies in DNA repair/damage response pathways impact on formation and function of male germ cells. We have investigated mice with deletions of ERCC1 (excision repair cross-complementing gene 1), MSH2 (MutS homolog 2, involved in mismatch repair pathway), and p53 (tumour suppressor gene implicated in elimination of germ cells with DNA damage).We demonstrate for the first time that depletion of ERCC1 or p53 from germ cells results in an increased incidence of unrepaired DNA breaks in pachytene spermatocytes and increased numbers of caspase-3 positive (apoptotic) germ cells. Sertoli cell-only tubules were detected in testes from mice lacking expression of ERCC1 or MSH2 but not p53. The number of sperm recovered from epididymes was significantly reduced in mice lacking testicular ERCC1 and 40% of sperm contained DNA breaks whereas the numbers of sperm were not different to controls in adult Msh2 -/- or p53 -/- mice nor did they have significantly compromised DNA.These data have demonstrated that deletion of Ercc1, Msh2 and p53 can have differential but overlapping affects on germ cell function and sperm production. These findings increase our understanding of the ways in which gene mutations can have an impact on male fertility

Angiotensin II-inhibiting drugs have no effect on intraneuronal Aβ or oligomeric Aβ levels in a triple transgenic mouse model of Alzheimer's disease

Background: Reducing the excessive accumulation of amyloid β-protein (Aβ) in Alzheimer's disease (AD) is a key objective of most AD therapies. Several studies suggest that pharmacological inhibition of angiotensin-converting enzyme (ACE) or its by-product angiotensin II may delay onset or progression of dementia and it has been suggested that this occurs via regulation of Aβ. Intraneuronal oligomeric accumulation of Aβ is postulated to be one of the earliest pathological events. Thus this study investigated the effect of an ACE-inhibitor, captopril, and two angiotensin II receptor blockers (ARBs), eprosartan and valsartan, on intraneuronal Aβ pathology and oligomeric Aβ levels in a triple transgenic (3xTGAD) mouse model of AD. Methods: Male, adult (3-4 month old) 3xTgAD mice (n=39) were randomly assigned to 4 treatment groups: valsartan (0.17g/l), eprosartan (0.8g/l), captopril (5g/l) or normal drinking water and the drugs given ad libitum for 2 months. Mean arterial blood pressure (MABP) was measured at baseline, at 2 weeks and at 2 months when the mice were sacrificed and the brains hemisected for analysis. One hemisphere was processed for Aβ and amyloid precursor protein (APP) immunohistochemistry and the other for biochemical measurement of oligomeric Aβ and APP. ACE activity was measured in the brain and kidney. Results: MABP was significantly reduced at 2 weeks and 2 months in the ACE-I group (p=0.0006) but was unaltered in the ARB groups compared to vehicle. Neither ACE-I nor ARB treatment altered Aβ and APP immunolabelling or the level of Aβ or APP in brain tissue homogenates. Similarly neither ACE-I nor ARB treatment altered ACE activity in either brain or kidney compared to control tissue. Conclusions: ACE-I or ARB administration over 2 months did not affect APP levels or either intraneuronal Aβ or oligomeric Aβ levels in 3xTGAD mice. While ARBs did not alter MABP, captopril did mediate reductions in MABP in the 3xTGAD mice which appeared to be independent of ACE activity. Further studies are needed to examine the effects of these drugs over a longer term and in older mice (i.e. when AD-like changes are more pronounced)

Susceptibility of epididymal sperm to acid denaturation detected by the SCSA.

<p>%DFI represents ratio of denatured sperm DNA (red) to total sperm DNA (red/[red+green]) fluorescence. ** (P<0.01) indicates significant variation compared with wild type littermates. For all three lines n = 4.</p

Proteomic analysis of mitochondria in APOE transgenic mice and in response to an ischemic challenge

Apolipoprotein E (APOE)-ɛ4 is associated with a deleterious outcome after ischemic brain injury, which may involve abnormal regulation of mitochondrial function. We have assessed the mitochondrial proteomic response of APOE-ɛ3 and APOE-ɛ4 transgenic mice to transient global ischemic injury in the hippocampus. A genotype-dependent increase in ApoE levels in mitochondria was observed after ischemia, with APOE-ɛ4 mice showing significantly greater increases than APOE-ɛ3 mice. Quantitative analysis of the mitochondria-enriched fractions was performed using liquid-chromatography mass spectrometry coupled to label-free analysis. Of the 1,067 identified proteins, 274 were mitochondria associated. Mitochondrial protein expression was significantly different between genotypes under basal conditions as well as in response to global ischemia. A total of 12 mitochondrial proteins (including respiratory chain proteins NDUFA11, NDUFS3, NDUF5B, ATP5J, as well as ETFA, CYB5B, ATP6V1A, HSPA1B, OXR1, GLUL, IARS2, and PHYHIPL) were significantly altered with respect to genotype, global ischemia, or their interaction (P<0.01). A compelling interactome, created using proteins found to be significantly modulated by global ischemia (P<0.05), involved proteins that regulate energy production and oxidative stress. Thus, APOE genotype has a differential effect on the mitochondrial protein expression in the absence and presence of an injury, which may underlie the differing genotype susceptibility

Summary of phenotypes of knockout mice and proposed role of proteins during spermatogenesis

<p>Summary of phenotypes of knockout mice and proposed role of proteins during spermatogenesis</p

Double strand breaks in pachytene spermatocytes detected using γH2AX immunohistochemistry on germ cell spreads.

<p>Immunodetection of SCP3 (red) and γH2AX (green) in <i>TG-Ercc1</i> (A–C), <i>Msh2</i> (E–G) and <i>p53</i> (I–K) spermatocytes. The Sex body is highlighted with white arrows and foci are highlighted with arrowheads. Quantification of γH2AX foci per 50 pachytene spermatocytes per mouse in D) <i>TG-Ercc1</i>, H) <i>Msh2</i> and L) <i>p53</i> (n = 3–7). ** (P<0.01) and *** (P<0.001) indicates significant variation compared to wild type.</p

Quantification of caspase-3 positive germ cells.

<p>The total number of caspase 3 positive cells was determined in 4 sections from each mouse. An * (P<0.05) indicates significant variation compared with wild type littermates. For <i>TG-Ercc1</i> data n = 5, for <i>Msh2</i> and <i>p53</i> data n = 3.</p

Histological evaluation of testes from adult mice.

<p>Haematoxylin and eosin staining of <i>Ercc1</i> (A–C), <i>Msh2</i> (E–G) and <i>p53</i> (I–K) testes. Sertoli cell only (SCO) tubules are highlighted with asterisks and gaps in testicular epithelium with arrows in <i>TG-Ercc1</i> −/− (C) and <i>Msh2</i> −/− (G). Bar = 100 µm. Distribution of seminiferous tubule diameters in D) <i>Ercc1</i>, H) <i>Msh2</i> and L) <i>p53</i> lines.</p