Homozygous Females Research Articles

Glucose-6-phosphate dehydrogenase deficiency A- variant in febrile patients in Haiti.

Haiti is one of two remaining malaria-endemic countries in the Caribbean. To decrease malaria transmission in Haiti, primaquine was recently added to the malaria treatment public health policy. One limitation of primaquine is that, at certain doses, primaquine can cause hemolytic anemia in individuals with glucose-6-phosphate dehydrogenase (G6PD) deficiency (G6PDd). In this study, we genotyped two mutations (A376G and G202A), which confer the most common G6PDd variant in West African populations, G6PDd A-. We estimated the frequency of G6PDd A- in a sample of febrile patients enrolled in an on-going malaria study who represent a potential target population for a primaquine mass drug administration. We found that 33 of 168 individuals carried the G6PDd A- allele (includes A- hemizygous males, A- homozygous or heterozygous females) and could experience toxicity if treated with primaquine. These data inform discussions on safe and effective primaquine dosing and future malaria elimination strategies for Haiti.

The RNA-binding protein, ZFP36L2, influences ovulation and oocyte maturation.

ZFP36L2 protein destabilizes AU-rich element-containing transcripts and has been implicated in female fertility. In the C57BL/6NTac mouse, a mutation in Zfp36l2 that results in the decreased expression of a form of ZFP36L2 in which the 29 N-terminal amino acid residues have been deleted, ΔN-ZFP36L2, leads to fertilized eggs that arrest at the two-cell stage. Interestingly, homozygous ΔN-Zfp36l2 females in the C57BL/6NTac strain release 40% fewer eggs than the WT littermates (Ramos et al., 2004), suggesting an additional defect in ovulation and/or oocyte maturation. Curiously, the same ΔN-Zfp36l2 mutation into the SV129 strain resulted in anovulation, prompting us to investigate a potential problem in ovulation and oocyte maturation. Remarkably, only 20% of ΔN-Zfp36l2 oocytes in the 129S6/SvEvTac strain matured ex vivo, suggesting a defect on the oocyte meiotic maturation process. Treatment of ΔN-Zfp36l2 oocytes with a PKA inhibitor partially rescued the meiotic arrested oocytes. Furthermore, cAMP levels were increased in ΔN-Zfp36l2 oocytes, linking the cAMP/PKA pathway and ΔN-Zfp36l2 with meiotic arrest. Since ovulation and oocyte maturation are both triggered by LHR signaling, the downstream pathway was investigated. Adenylyl cyclase activity was increased in ΔN-Zfp36l2 ovaries only upon LH stimulation. Moreover, we discovered that ZFP36L2 interacts with the 3′UTR of LHR mRNA and that decreased expression levels of Zfp36l2 correlates with higher levels of LHR mRNA in synchronized ovaries. Furthermore, overexpression of ZFP36L2 decreases the endogenous expression of LHR mRNA in a cell line. Therefore, we propose that lack of the physiological down regulation of LHR mRNA levels by ZFP36L2 in the ovaries is associated with anovulation and oocyte meiotic arrest.

The Drosophila Su(var)3-7 gene is required for oogenesis and female fertility, genetically interacts with piwi and aubergine, but impacts only weakly transposon silencing.

Heterochromatin is made of repetitive sequences, mainly transposable elements (TEs), the regulation of which is critical for genome stability. We have analyzed the role of the heterochromatin-associated Su(var)3–7 protein in Drosophila ovaries. We present evidences that Su(var)3–7 is required for correct oogenesis and female fertility. It accumulates in heterochromatic domains of ovarian germline and somatic cells nuclei, where it co-localizes with HP1. Homozygous mutant females display ovaries with frequent degenerating egg-chambers. Absence of Su(var)3–7 in embryos leads to defects in meiosis and first mitotic divisions due to chromatin fragmentation or chromosome loss, showing that Su(var)3–7 is required for genome integrity. Females homozygous for Su(var)3–7 mutations strongly impair repression of P-transposable element induced gonadal dysgenesis but have minor effects on other TEs. Su(var)3–7 mutations reduce piRNA cluster transcription and slightly impact ovarian piRNA production. However, this modest piRNA reduction does not correlate with transposon de-silencing, suggesting that the moderate effect of Su(var)3–7 on some TE repression is not linked to piRNA production. Strikingly, Su(var)3–7 genetically interacts with the piwi and aubergine genes, key components of the piRNA pathway, by strongly impacting female fertility without impairing transposon silencing. These results lead us to propose that the interaction between Su(var)3–7 and piwi or aubergine controls important developmental processes independently of transposon silencing.

Response to Zhang et al

Two recent reports by Zhang et al published in Nov (2011) and our group (Anand & Kai, 2012; published online in Dec 2011) have suggested a role for the conserved Tudor domain protein encoded by qin as a component of piRNA pathway. Both studies agree that qin plays a role in the piRNA pathway and is required to repress reteroelements but differ in some aspects of their conclusions about qin function. We reported that kumo M41‐13 , hereafter designated qin kumo , homozygous females exhibited mislocalization of many piRNA pathway components from the nuage, a perinuclear structure where piRNA pathway components are concentrated in the ovary. We also reported a reduction in piRNAs targeting transposons (Supplementary Table 3 in Anand & Kai, 2012). In contrast, Zhang et al found no change in localization of piRNA pathway components to the nuage and observed an increase in Aubergine(Aub)‐Aub homotypic ping‐pong in the qin 1 /Df alleleic combination, which led Zhang et al to propose that qin is required for maintenance of heterotypic, Aub‐Argonaute3 (Ago3) ping‐pong (Zhang et al , 2011). In the accompanying Correspondence, Zhang et al challenge some of our findings. The key issue is whether the localization of piRNA pathway proteins to the nuage is compromised in absence of qin . The two groups used different alleles …

Subfertility and growth restriction in a new galactose-1 phosphate uridylyltransferase (GALT) - deficient mouse model

The first GalT gene knockout (KO) mouse model for Classic Galactosemia (OMIM 230400) accumulated some galactose and its metabolites upon galactose challenge, but was seemingly fertile and symptom free. Here we constructed a new GalT gene-trapped mouse model by injecting GalT gene-trapped mouse embryonic stem cells into blastocysts, which were later implanted into pseudo-pregnant females. High percentage GalT gene-trapped chimera obtained were used to generate heterozygous and subsequently, homozygous GalT gene-trapped mice. Biochemical assays confirmed total absence of galactose-1 phosphate uridylyltransferase (GALT) activity in the homozygotes. Although the homozygous GalT gene-trapped females could conceive and give birth when fed with normal chow, they had smaller litter size (P=0.02) and longer time-to-pregnancy (P=0.013) than their wild-type littermates. Follicle-stimulating hormone levels of the mutant female mice were not significantly different from the age-matched, wild-type females, but histological examination of the ovaries revealed fewer follicles in the homozygous mutants (P=0.007). Administration of a high-galactose (40% w/w) diet to lactating homozygous GalT gene-trapped females led to lethality in over 70% of the homozygous GalT gene-trapped pups before weaning. Cerebral edema, abnormal changes in the Purkinje and the outer granular cell layers of the cerebellum, as well as lower blood GSH/GSSG ratio were identified in the galactose-intoxicated pups. Finally, reduced growth was observed in GalT gene-trapped pups fed with normal chow and all pups fed with high-galactose (20% w/w) diet. This new mouse model presents several of the complications of Classic Galactosemia and will be useful to investigate pathogenesis and new therapies.

Development of a Mouse Model of Menopausal Ovarian Cancer

Despite significant understanding of the genetic mutations involved in ovarian epithelial cancer and advances in genomic approaches for expression and mutation profiling of tumor tissues, several key questions in ovarian cancer biology remain enigmatic: the mechanism for the well-established impact of reproductive factors on ovarian cancer risk remains obscure; cell of origin of ovarian cancer continue to be debated; and the precursor lesion, sequence, or events in progression remain to be defined. Suitable mouse models should complement the analysis of human tumor tissues and may provide clues to these questions currently perplexing ovarian cancer biology. A potentially useful model is the germ cell-deficient Wv (white spotting variant) mutant mouse line, which may be used to study the impact of menopausal physiology on the increased risk of ovarian cancer. The Wv mice harbor a point mutation in c-Kit that reduces the receptor tyrosine kinase activity to about 1–5% (it is not a null mutation). Homozygous Wv mutant females have a reduced ovarian germ cell reservoir at birth and the follicles are rapidly depleted upon reaching reproductive maturity, but other biological phenotypes are minimal and the mice have a normal life span. The loss of ovarian function precipitates changes in hormonal and metabolic activity that model features of menopause in humans. As a consequence of follicle depletion, the Wv ovaries develop ovarian tubular adenomas, a benign epithelial tumor corresponding to surface epithelial invaginations and papillomatosis that mark human ovarian aging. Ongoing work will test the possibility of converting the benign epithelial tubular adenomas into neoplastic tumors by addition of an oncogenic mutation, such as of Tp53, to model the genotype and biology of serous ovarian cancer. Model based on the Wv mice may have the potential to gain biological and etiological insights into ovarian cancer development and prevention.

Multiple paternity in wild house mice (Mus musculus musculus): effects on offspring genetic diversity and body mass

Multiple mating is common in many species, but it is unclear whether multiple paternity enhances offspring genetic diversity or fitness. We conducted a survey on wild house mice (Mus musculus musculus), and we found that in 73 pregnant females, 29% of litters had multiple sires, which is remarkably similar to the 23–26% found in feral populations of Mus musculus domesticus in the USA and Australia, respectively. The question is: How has selection maintained multiple mating in these subspecies since the evolutionary divergence, ca. 2800–6000 years ago? We found no evidence that multiple paternity enhanced females’ litter size, contrary to the fertility assurance or genetic benefits hypotheses. Multiple paternity was associated with reduced mean and variance in offspring body mass, which suggests that females allocate fewer resources or that there is increased intrauterine conflict in multiple-versus single-sired litters. We found increased allelic diversity (though not heterozygosity) in multiple-sired litters, as predicted by the genetic diversity hypothesis. Finally, we found that the dams’ heterozygosity was correlated with the mean heterozygosity of their offspring in single-and multiple-sired litters, suggesting that outbred, heterozygous females were more likely to avoid inbreeding than inbred, homozygous females. Future studies are needed to examine how increased genetic diversity of litters and smaller mean (and variance) offspring body mass associated with multiple paternity affect offspring fitness.

COL4A4-related nephropathy caused by a novel mutation in a large consanguineous Saudi family

IntroductionCollagen type IV related nephropathies are due to the defects in collagen IV genes COL4A3, COL4A4, or COL4A5 and comprise a spectrum of phenotypes ranging from Alport Syndrome (AS) to its mild variants, termed as familial haematuria or thin basement membrane nephropathy. Classical AS is a progressive renal disease presenting with a triad of progressive hematuric nephritis and typical extra-renal complications, such as sensorineural hearing loss (SNHL) and variable ocular anomalies. The mode of inheritance in AS is X-linked in 85%, autosomal recessive in 15%, and autosomal dominant in rare cases. ObjectivesThis study aims to identify underlying mutation in multiple individuals from a large consanguineous Saudi family with inherited nephropathy, including our index patient who manifested all the features of classical AS. Patients and methodsPatients were diagnosed by nephrologists and clinical geneticists. All the individuals underwent clinical, audiological and ophthalmological evaluation. Blood samples were collected after written informed consent. DNA extraction, homozygosity mapping and PCR amplification followed standard methodologies. ResultsThe disease locus was mapped to 2q36.3, where both COL4A3 and COL4A4 reside. Sanger sequencing of COL4A3 and COL4A4 revealed an underlying novel homozygous disease-causing COL4A4 mutation (c.2420delG; p.G807fsX60) in the affected proband. Considerable phenotypic variability segregating with this COL4A4 mutation in our study family is documented. The homozygous mutants were manifesting end-stage renal disease (ESRD) in their adolescence, while the heterozygous carrier members were presenting with considerable phenotypic heterogeneity ranging from intermittent hematuria to late onset ESRD. In addition, there is a relatively severe involvement of the ear (SNHL) and eye in the homozygotes than the heterozygotes. Fertility problems were also noted in both of the homozygous females. ConclusionIdentification of the causative mutation is an efficient strategy for conclusive molecular diagnosis in the patients and to establish genotype/phenotype correlation. It is important to study and evaluate asymptomatic carriers, to predict prognosis of the disease and to obviate the need for another renal biopsy in at-risk related family members. While an accurate genetic diagnosis of AS provides valuable information for genetic counseling in the extended family members, it can also facilitate future prenatal diagnosis and planning for pre-implantation genetic diagnosis.

Genetic Effects on Cerebellar Structure Across Mouse Models of Autism Using a Magnetic Resonance Imaging Atlas

Magnetic resonance imaging (MRI) of autism populations is confounded by the inherent heterogeneity in the individuals' genetics and environment, two factors difficult to control for. Imaging genetic animal models that recapitulate a mutation associated with autism quantify the impact of genetics on brain morphology and mitigate the confounding factors in human studies. Here, we used MRI to image three genetic mouse models with single mutations implicated in autism: Neuroligin-3 R451C knock-in, Methyl-CpG binding protein-2 (MECP2) 308-truncation and integrin β3 homozygous knockout. This study identified the morphological differences specific to the cerebellum, a structure repeatedly linked to autism in human neuroimaging and postmortem studies. To accomplish a comparative analysis, a segmented cerebellum template was created and used to segment each study image. This template delineated 39 different cerebellar structures. For Neuroligin-3 R451C male mutants, the gray (effect size (ES) = 1.94, FDR q = 0.03) and white (ES = 1.84, q = 0.037) matter of crus II lobule and the gray matter of the paraflocculus (ES = 1.45, q = 0.045) were larger in volume. The MECP2 mutant mice had cerebellar volume changes that increased in scope depending on the genotype: hemizygous males to homozygous females. The integrin β3 mutant mouse had a drastically smaller cerebellum than controls with 28 out of 39 cerebellar structures smaller. These imaging results are discussed in relation to repetitive behaviors, sociability, and learning in the context of autism. This work further illuminates the cerebellum's role in autism.

5-HTTLPR, anxiety and gender interaction moderates right amygdala volume in healthy subjects

Genetic variants within the serotonin transporter gene (5-HTTLPR) impact the neurobiology and risk for anxiety-related behaviours. There are also gender differences in the prevalence of anxiety-related behaviours. Although numerous studies have investigated the influence of 5-HTTLPR genotype on the neural systems involved in emotional regulation, none have investigated how these effects are modulated by gender and anxiety. We investigated this issue using two complementary region of interest-based structural neuroimaging approaches (voxel-based morphometry and Freesurfer) in 138 healthy individuals categorized into 'no anxiety' and 'subclinical anxiety' groups based on the Hamilton Rating Scale for Anxiety (HAM-A). Preliminarily, using anxiety as a continuous variable, we found a significant interaction effect of genotype by gender on anxiety. Females homozygous for the Short allele showed the highest HAM-A scores and males the lowest. In addition, a three-way significant interaction among genotype, gender and anxiety category was found for the right amygdala volume. Post hoc tests revealed that homozygous females carrying the Short variant with a subclinical anxiety condition had larger volume. The reported interaction effects demonstrate that gender strongly modulates the relationship between 5-HTTLPR genotype and subclinical expression of anxiety acting on amygdala, one region of the emotional neural network specifically involved in the anxiety-like behaviours.

A Selfish Genetic Element Influencing Longevity Correlates with Reactive Behavioural Traits in Female House Mice (Mus domesticus)

According to theory in life-history and animal personality, individuals with high fitness expectations should be risk-averse, while individuals with low fitness expectations should be more bold. In female house mice, a selfish genetic element, the t haplotype, is associated with increased longevity under natural conditions, representing an appropriate case study to investigate this recent theory empirically. Following theory, females heterozygous for the t haplotype (+/t) are hypothesised to express more reactive personality traits and be more shy, less explorative and less active compared to the shorter-lived homozygous wildtype females (+/+). As males of different haplotype do not differ in survival, no similar pattern is expected. We tested these predictions by quantifying boldness, exploration, activity, and energetic intake in both +/t and +/+ mice. +/t females, unlike +/+ ones, expressed some reactive-like personality traits: +/t females were less active, less prone to form an exploratory routine and tended to ingest less food. Taken together these results suggest that differences in animal personality may contribute to the survival advantage observed in +/t females but fail to provide full empirical support for recent theory.

PWE-136 The Effect of C282Y Homozygosity on Full Blood Count Indices

IntroductionFull blood count (FBC) indices and iron studies are utilised in initial screening of patients with clinical suspicion of haemachromatosis and HFE genetic testing is used for diagnosis. The aim...

Long-Standing Balancing Selection in theTHBS4Gene: Influence on Sex-Specific Brain Expression and Gray Matter Volumes in Alzheimer Disease

The THBS4 gene encodes a glycoprotein involved in inflammatory responses and synaptogenesis. THBS4 is expressed at higher levels in the brain of humans compared with nonhuman primates, and the protein accumulates in β-amyloid plaques. We analyzed THBS4 genetic variability in humans and show that two haplotypes (hap1 and hap2) are maintained by balancing selection and modulate THBS4 expression in lymphocytes. Indeed, the balancing selection region covers a predicted transcriptional enhancer. In humans, but not in macaques and chimpanzees, THBS4 brain expression increases with age, and variants in the balancing selection region interact with sex in influencing THBS4 expression (pinteraction = 0.038), with hap1 homozygous females showing lowest expression. In Alzheimer disease (AD) patients, significant interactions between sex and THBS4 genotype were detected for peripheral gray matter (pinteraction = 0.014) and total gray matter (pinteraction = 0.012) volumes. Similarly to the gene expression results, the interaction is mainly mediated by hap1 homozygous AD females, who show reduced volumes. Thus, the balancing selection target in THBS4 is likely represented by one or more variants that regulate tissue-specific and sex-specific gene expression. The selection signature associated with THBS4 might not be related to AD pathogenesis, but rather to inflammatory responses.

Glucose-6-phosphate dehydrogenase deficiency in Tunisia: molecular data and phenotype-genotype association

Glucose-6-phosphate dehydrogenase (G6PD) deficiency is the most common human enzyme defect. In this study, we aimed to perform a molecular investigation of G6PD deficiency in Tunisia and to associate clinical manifestations and the degree of deficiency with the genotype. A total of 161 Tunisian subjects of both sexes were screened by spectrophotometric assay for enzyme activity. Out of these, 54 unrelated subjects were selected for screening of the most frequent mutations in Tunisia by PCR/RFLP, followed by size-based separation of double-stranded fragments under non-denaturing conditions on a denaturing high performance liquid chromatography system. Of the 56 altered chromosomes examined, 75% had the GdA(-) mutation, 14.28% showed the GdB(-) mutation and no mutations were identified in 10.72% of cases. Hemizygous males with GdA(-) mutation were mostly of class III, while those with GdB(-) mutation were mainly of class II. The principal clinical manifestation encountered was favism. Acute hemolytic crises induced by drugs or infections and neonatal jaundice were also noted. Less severe clinical features such as low back pain were present in heterozygous females and in one homozygous female. Asymptomatic individuals were in majority heterozygote females and strangely one hemizygous male. The spectrum of mutations seems to be homogeneous and similar to that of Mediterranean countries; nevertheless 10.72% of cases remain with undetermined mutation thus suggesting a potential heterogeneity of the deficiency at the molecular level. On the other hand, we note a better association of the molecular defects with the severity of the deficiency than with clinical manifestations.

Homozygous factor V Leiden and double heterozygosity for factor V Leiden and prothrombin mutation

The most common forms of familial thrombophilia are factor V Leiden (FVL) and prothrombin mutation (PTM). Homozygous FVL and PTM have long been feared conditions thought to cause high rates of morbidity and mortality. To analyse clinical features in patients with homozygous FVL and PTM, as well as patients with double heterozygosity for FVL and PTM. All patients with homozygous FVL, PTM or double heterozygosity in the MATS database of 1465 consecutive unselected patients were analysed regarding age at inclusion venous thromboembolism (VTE), age at first thrombosis, recurrence, clinical course and acquired risk factors. We found 36 patients homozygous for FVL. Patients homozygous for FVL were younger than controls at group level (56±18 vs. 63±17, p<0.02). Homozygous women were younger than female controls (50±19 vs. 63±18, p<0.002). No difference was observed when comparing male subjects. Women were younger than men at inclusion thrombosis (50±19 vs. 65±14, p<0.02) and at first thrombosis (47±19 vs. 64±14, p<0.01). Deep venous thrombosis (DVT) was seen in 33 patients (92%), 6 (17%) had pulmonary embolism (PE) and 3 (8%) had combined DVT and PE. PE was less frequent in homozygous FVL women compared to female controls (p<0.03). VTE recurred in 3 subjects during the duration of the study. Odds ratio for VTE in homozygous FVL patients compared to controls was 13.9 (95% CI 9.9-19.7). We found no subjects with homozygous PTM. Double heterozygosity for FVL and PTM was seen in 12 subjects. There was no difference in age at inclusion VTE between double heterozygotes and controls (59±16 vs. 63±17, ns.). DVT was seen in 92% at inclusion, 8% had PE. Mean age at first VTE was 52±17 (27-82). Consecutive homozygous FVL patients had a higher age at first thrombosis than previously described. Homozygous females are affected at an earlier age than homozygous men and female controls. It seems that thrombi in homozygous FVL have a different pattern compared to controls i.e. more prone for thrombosis in the lower extremity. The odds ratio for thrombosis among homozygous FVL seems to be lower than previously described.

Genetic sex separation of the malaria vector, Anopheles arabiensis, by exposing eggs to dieldrin

BackgroundThe sterile insect technique (SIT) has been used with success for suppressing or eliminating important insect pests of agricultural or veterinary importance. In order to develop SIT for mosquitoes, female elimination prior to release is essential as they are the disease-transmitting sex. A genetic sexing strain (GSS) of Anopheles arabiensis was created based on resistance to dieldrin, and methods of sex separation at the egg stage were developed. The use of this strain for SIT will require sexually sterile males: useful radiation doses for this purpose were determined for pupae and adults.MethodsFor the creation of the sexing strain, dieldrin-resistant males were irradiated with 40 Gy using a 60Co source and were subsequently crossed to homozygous susceptible virgin females. Individual families were screened for semi-sterility and for male resistance to dieldrin. For sex separation, eggs of a resulting GSS, ANO IPCL1, were exposed to varying concentrations of dieldrin for different durations. Percent hatch, larval survival, and male and female emergence were recorded. Radiation induced sterility was determined following adult and pupa exposure to gamma rays at 0–105 Gy. Mortality induced by dieldrin treatment, and levels of sterility post radiation were investigated.ResultsANO IPCL1 contains a complex chromosome aberration that pseudo-links the male-determining Y chromosome and dieldrin resistance, conferring high natural semi-sterility. Exposure of eggs to 2, 3, and 4 ppm dieldrin solutions resulted in complete female elimination without a significant decrease of male emergence compared to the controls. A dose of 75 Gy reduced the fertility to 3.8 and 6.9% when males were irradiated as pupae or adults respectively, but the proportions of progeny of these males reaching adulthood were 0.6 and 1.5% respectivelyConclusionThe GSS ANO IPCL1 was shown to be a suitable strain for further testing for SIT though high semi-sterility is a disadvantage for mass rearing.

Associations between single nucleotide polymorphisms of GDF9 and BMP15 genes and litter size in two dairy sheep breeds of Greece

The aim of the present study was to assess the presence of single nucleotide polymorphisms (SNPs) in the Growth Differentiating Factor 9 (GDF9) and Bone Morphogenetic Protein 15 (BMP15) genes, in two Greek sheep breeds i.e. Chios and Karagouniki, with high and moderate litter size (LS) i.e. number of lambs born, respectively. An examination of association(s) between the polymorphisms and LS, within breeds, was also conducted. Blood samples from 92 Chios and 96 Karagouniki ewes were collected, while repeated records on LS, were also available (n=239 and 259, respectively). Detection of SNPs was performed on the DNA products by PCR-RLFP analysis. Data analysis included examination of allelic and genotypic differentiation between the breeds, testing for Hardy–Weinberg Equilibrium (HWE) and association analysis between polymorphisms and LS. Genetic analysis showed that the G1 and G8 mutations of the GDF9 gene were significantly over-presented only in the highly prolific breed (Chios). The B4 mutation of the BMP15 gene was significantly over-presented only in the low prolific breed (Karagouniki). Both breeds were in Hardy–Weinberg disequilibrium (HWD), possibly due to selection pressure, population sub-structure and genetic drift. Certain polymorphisms of the G1, G4 and G8 sites of the GDF9 gene in the Chios breed were found to show heterozygote advantage with no evidence of infertility for the homozygous females. A similar trend was observed in the BMP15 gene although at no statistically significant level. In the Karagouniki breed, no significant associations between the studied polymorphisms and LS were detected.

Influence of the zona pellucida of the mouse egg on folliculogenesis and fertility

All mammalian eggs are surrounded by a relatively thick extracellular coat, called the zona pellucida (ZP), that plays vital roles during oogenesis, fertilization, and preimplantation development. The mouse egg ZP consists of three glycoproteins, called mZP1-3, that are synthesized solely by oocytes during their 2-to-3 week growth phase. The ZP is seen initially as isolated extracellular deposits of nascent ZP fibrils that coalesce to form a thickening matrix. Elimination of ZP glycoprotein synthesis by targeted mutagenesis yields mice that are heterozygous or homozygous for the null mutations. Homozygous null males are unaffected by the mutations and heterozygous females are as fertile as wild-type females. On the other hand, eggs from mZP2-/- and mZP3-/- females lack a ZP and the mice are completely infertile due to a severely reduced number of ovulated eggs in their oviducts. Development of ovarian follicles is retarded in homozygous null females and manifested as reduced ovarian weight, number of Graafian follicles, and number of ovulated eggs. Eggs from mZP1-/- females have a distorted ZP and, although the mice are fertile, they exhibit reduced fertility due to early embryonic loss. Potential relationships between ZP biogenesis, folliculogenesis, and fertility are addressed.

Role of Moesin in Murine Hematopoiesis

Abstract 1338 Background:Moesin, a membrane-cytoskeleton linker protein, functions in microtubule formation, in membrane ruffling, and at sites of cell-adhesion. However, its role in hematopoiesis is unclear. Although a previous study demonstrated normal hematopoietic function in moesin knock-out (KO) mice (JBC 1999), the changes in blood cell counts observed in the mice have not been thoroughly analyzed. Moesin-specific antibodies are detected in the serum of approximately 30% of patients with acquired aplastic anemia (AA). Moesin is expressed on the surface of peripheral blood T cells and monocytes, but not on hematopoietic stem cells (HSCs). Our recent study demonstrated the involvement of anti-moesin antibodies in the development of AA by stimulating T cells and monocytes to secrete tumor necrosis factor-α and interferon-γ. Given that hematopoiesis is regulated by T cells and monocytes, the absence of moesin should impact hematopoiesis. The purpose of this study was to examine blood cell counts in moesin KO mice and WT mice transplanted with anti-moesin antibody producing B cells to clarify the role of moesin in hematopoiesis. Methods:Moesin KO mice (F1 heterozygous females (-/X)), which were a hybrid strain between 129/Sv and C57BL/6 (B6), were kindly provided by Professor Tsukita of Osaka University. F1 heterozygous females and B6 F1 wild-type (WT) males were interbred and the littermates were genotyped. Moesin KO mice, F2 hemizygous males (-/Y), and F3 homozygous females (−/−) were analyzed in a previous study (JBC 1999). F1 heterozygous females were backcrossed to B6 males for at least 8 generations to generate moesin KO mice in a B6 background. Moesin KO mice (12 months old) were used for hematopoietic phenotype analysis and to raise anti-moesin antibodies by immunization with moesin protein. Moesin KO male mice (-/Y) were immunized with 2×107 spleen cells containing moesin from WT B6 male mice to produce anti-moesin antibody secreting cells. Anti-moesin antibody titers were determined by Western blot analysis. To analyze the effects of anti-moesin antibodies in hematopoiesis, spleen cells (1.5×107) and bone marrow (BM) cells (1×107) containing anti-moesin antibody secreting cells from moesin KO mice were transplanted to the BM of 8-week-old WT B6 male mice conditioned with irradiation (3 Gy). Results:Moesin KO mice were born at expected Mendelian ratios and developed and grew normally. However, the body weight of moesin KO mice (n=7) was lower than that of WT mice (n=9) (26.63±2.47 g vs. 30.71±4.85 g; P=0.049). Complete blood counts of moesin KO mice (n=4) revealed leukocytopenia (WBC 12000±3096/μL vs. 19750±2582/μL; P=0.03) and macrocytic anemia (Hb 13.8±0.50 g/dL vs. 15.8±0.50 g/dL; P=0.06; MCV 51.7±1.7 fL vs. 47.6±1.3 fL, P=0.002) as compared with WT mice (n=4); platelet counts were comparable. Moesin KO mouse spleens (n=8) were heavier than those of WT mice (0.316±0.355 g vs. 0.091±0.034 g; P=0.0002) and follicle formation in spleens of moesin KO mice were obscure with increased CD3+ lymphocytes and reduced CD19+ lymphocytes as compared with WT mice. BM of moesin KO mice was normocellular and characterized by increased CD3+ lymphocytes and reduced CD19+ lymphocytes as seen in spleens of moesin KO mice. The number of lineage negative, Sca-1+, and c-Kit+ (LSK) mouse HSCs in moesin KO BM (n=4) tended to be lower than that of WT mice (n=3) (0.97±0.29% vs. 1.85±0.20%; P=0.06). Moesin KO spleen/BM cells capable of producing anti-moesin antibodies were infused into BM of WT B6 male mice (n=3), and moesin KO hematopoietic cells were engrafted on day 21. Transplanted mice showed leukocytopenia compared to WT B6 male mice transplanted with WT spleen/BM cells (n=3) (WBC 2667±333/μL vs. 7333±333/μL; P=0.0006), but anemia and thrombocytopenia were not observed. Conclusions:Moesin plays an important role in murine hematopoietic and immune system development. Anti-moesin antibody-induced leukocytopenia may result from excessive secretion of myelosuppressive cytokines from immune cells as observed in AA patients. The mechanistic details of HSC impairment in moesin KO mice are currently under investigation. Disclosures:No relevant conflicts of interest to declare.

Isolation and Genetic Characterization of Mother-of-Snow-White, a Maternal Effect Allele Affecting Laterality and Lateralized Behaviors in Zebrafish

In the present work we report evidence compatible with a maternal effect allele affecting left-right development and functional lateralization in vertebrates. Our study demonstrates that the increased frequency of reversed brain asymmetries in a zebrafish line isolated through a behavioral assay is due to selection of mother-of-snow-white (msw), a maternal effect allele involved in early stages of left-right development in zebrafish. msw homozygous females could be identified by screening of their progeny for the position of the parapineal organ because in about 50% of their offspring we found an altered, either bilateral or right-sided, expression of lefty1 and spaw. Deeper investigations at earlier stages of development revealed that msw is involved in the specification and differentiation of precursors of the Kupffer's vesicle, a structure homologous to the mammalian node. To test the hypothesis that msw, by controlling Kupffer's vesicle morphogenesis, controls lateralized behaviors related to diencephalic asymmetries, we analyzed left- and right-parapineal offspring in a “viewing test”. As a result, left- and right-parapineal individuals showed opposite and complementary eye preference when scrutinizing a model predator, and a different degree of lateralization when scrutinizing a virtual companion. As maternal effect genes are expected to evolve more rapidly when compared to zygotic ones, our results highlight the driving force of maternal effect alleles in the evolution of vertebrates behaviors.