KATNAL1 regulation of sertoli cell microtubule dynamics is essential for spermiogenesis and male fertility.

Spermatogenesis is a complex process reliant upon interactions between germ cells (GC) and supporting somatic cells. Testicular Sertoli cells (SC) support GCs during maturation through physical attachment, the provision of nutrients, and protection from immunological attack. This role is facilitated by an active cytoskeleton of parallel microtubule arrays that permit transport of nutrients to GCs, as well as translocation of spermatids through the seminiferous epithelium during maturation. It is well established that chemical perturbation of SC microtubule remodelling leads to premature GC exfoliation demonstrating that microtubule remodelling is an essential component of male fertility, yet the genes responsible for this process remain unknown. Using a random ENU mutagenesis approach, we have identified a novel mouse line displaying male-specific infertility, due to a point mutation in the highly conserved ATPase domain of the novel KATANIN p60-related microtubule severing protein Katanin p60 subunit A-like1 (KATNAL1). We demonstrate that Katnal1 is expressed in testicular Sertoli cells (SC) from 15.5 days post-coitum (dpc) and that, consistent with chemical disruption models, loss of function of KATNAL1 leads to male-specific infertility through disruption of SC microtubule dynamics and premature exfoliation of spermatids from the seminiferous epithelium. The identification of KATNAL1 as an essential regulator of male fertility provides a significant novel entry point into advancing our understanding of how SC microtubule dynamics promotes male fertility. Such information will have resonance both for future treatment of male fertility and the development of non-hormonal male contraceptives.

Collagen dysregulation in the dermis of the Sagg/+ mouse: a loose skin model.

The Sagg/+ mouse is an ethylnitrosourea-derived mutant with a dermal phenotype similar to some of the subtypes of Ehlers-Danlos syndrome (EDS) and cutis laxa. The dermis of the Sagg/+ mouse has less dense and more disorganized collagen fibers compared to controls. The size of extracted Type I dermal collagen was the same as that observed in normal skin; however, more collagen could be extracted from Sagg/+ skin, which also showed decreased collagen content and decreased steady-state levels of alpha1(I), alpha2(I), alpha1(V), and alpha2(V) procollagen mRNAs. The biomechanical properties of Sagg/+ skin were significantly decreased relative to normal skin. However, there were no significant differences in the quantities of the major collagen cross-links, that is, dehydrohydroxylysinonorleucine and dehydrohistidinohydroxymerodesmosine between Sagg/+ and normal skin. Electron microscopic evaluation of Sagg/+ skin indicated that the mutation interferes with the proper formation of collagen fibrils and the data are consistent with a mutation in Type V collagen leading to haploinsufficiency with the formation of two sub-populations of collagen fibrils, one normal and one with irregular shape and a larger diameter. Further study of this novel mutation will allow the identification of new mechanisms involved in the regulation of normal and pathologic collagen gene expression.

Progressive loss of motor neuron function in wasted mice: effects of a spontaneous null mutation in the gene for the eEF1 A2 translation factor.

Wasted (wst) is a spontaneous autosomal recessive mutation in which the gene encoding translation factor eEF1A2 is deleted. Homozygous mice show tremors and disturbances of gait shortly after weaning, followed by motor neuron degeneration, paralysis, and death by about 28 days. We have now conducted a more detailed analysis of neuromuscular pathology in these animals. Reactive gliosis was observed at 19 days postnatal in wst/wst cervical spinal cord, showing a rostrocaudal gradient. This was followed a few days later by motor neuron vacuolation and neurofilament accumulation, again with a rostrocaudal progression. Thoracic/abdominal muscles from wst/wst mice aged 17 days showed evidence of progressive denervation of motor endplates, including weak synaptic transmission and retraction of motor nerve terminals. Similar abnormalities appeared in distal, lumbrical muscles from about 25 days of age. We conclude that spontaneous failure of eEF1A2 expression in the wasted mutant first triggers gliosis in spinal cord and retraction of motor nerve terminals in muscle, and then motor neuron pathology and death. The early initiation and rapid progression of motor unit degeneration in wst/wst mice suggest that they should be considered an important and accessible model of early-onset motor neuron degeneration in humans.

Interactions between imprinting effects in the mouse.

Mice with uniparental partial or complete disomies for any one of 11 identified chromosomes show abnormal phenotypes. The abnormalities, or imprinting effects, can be attributable to an incorrect dosage of maternal or paternal copies of imprinted gene(s) located within the regions involved. Here we show that combinations of partial disomies may result in interactions between imprinting effects that seemingly independently affect fetal and/or placental growth in different ways or modify neonatal and postnatal imprinting effects. Candidate genes within the regions have been identified. The findings are generally in accord with the "conflict hypothesis" for the evolution of genomic imprinting but do not clearly demonstrate common growth axes within which imprinted genes may interact. Instead, it would seem that any gene that represses or limits embryonic/fetal growth to the advantage of the mother--by any developmental means--will have been subject to evolutionary selection for paternal allele repression. Likewise, any gene that favors embryonic/fetal development at consequent cost to the mother--by any developmental means--will have faced selection for maternal allele repression. The classical Igf2-Igf2r axis may therefore be unique. The findings involve reinterpretation of older imprinting data and consequently revision of the mouse imprinting map.

Demonstration of autoimmunity in the tight skin-2 mouse: a model for scleroderma.

The tight skin-2 (Tsk2/+) mouse has been proposed as an animal model of systemic sclerosis (SSc) because this animal exhibits increased collagen synthesis and accumulation in the dermis. The Tsk2/+ mouse also has been reported to have a mononuclear cell infiltrate in the dermis; however, to date no evidence of autoimmunity has been described in this animal model. We report here that Tsk2/+ mice harbor numerous autoantibodies in their plasma including some, which are similar to those, present in SSc patients. Immunofluorescence with HEp-2 cells revealed the presence of anti-nuclear Abs (ANAs) in the plasma of 92% of the Tsk2/+ mice. In contrast, <5% of cage-mated CAST/ei mice had a positive ANA and none of the C3H/HeJ age-matched controls were positive. Homogenous, speckled, rim, nucleolar, centromere as well as combinations of these patterns were observed. The proportion of Tsk2/+ animals with a positive ANA increased slightly with age. ELISAs showed that 93% of the Tsk2/+ animals were positive for anti-Scl70, 82% for anti-centromere, 5% for anti-RNP/Sm, and none were positive for anti-RNA-polymerase II Abs. Indirect immunofluorescence with Crithidia luciliae and ELISA for anti-dsDNA Abs showed that 76% of Tsk2/+ mice were positive for this autoantibody. The high frequency of anti-Scl70 and anti-centromere autoantibodies indicates that Tsk2/+ mice display some humoral immune alterations which are similar to those found in patients with SSc. However, the Tsk2/+ mice also develop autoantibodies to dsDNA and a majority of the mice develop multiple autoantibody specificities (anti-Scl70, anti-CENP-B, and anti-dsDNA) indicating that the mouse may be a useful model to study autoimmunity in a wider spectrum of connective tissue diseases.

Two ENU-induced mutations in Rasgrf1 and early mouse growth retardation.

When paternally transmitted, two independent ENU-induced mutations showed reduced whole body wet weight soon after birth. The mutations were mapped to Chromosome 9 (Chr 9) between the markers D9Mit208 and D9Mit215. Their map position and imprinted status suggested that they might alter RAS protein-specific guanine nucleotide releasing factor 1 expression. Both mutations introduced premature chain termination codons into the coding sequence of Rasgrf1, and no Ras-GRF1 protein was detected in the brain. The GENA53 line had a C to T transition at nucleotide 2137, and the line GENA37 had a T to A transversion at nucleotide 3552 of the cDNA sequence. Mutant mice had near normal body weight at birth, but their weight started to lag behind that of wild-type littermates during the first week, and they were about 15% lighter as adults.

Alternative non-coding splice variants of Nespas, an imprinted gene antisense to Nesp in the Gnas imprinting cluster.

The Gnas locus on mouse Chr 2 represents a unique cluster of overlapping imprinted genes. Three of these in the order Nesp--Gnasxl--Gnas are transcribed in the sense direction with Nesp having maternal-specific expression, Gnasxl having paternal expression, and Gnas as being biallelically expressed in most tissues. A fourth imprinted gene, Nespas, is paternally expressed, lies antisense to Nesp, and expresses an unspliced transcript. Large unspliced antisense transcripts are emerging as a feature of imprinted gene clusters, and such non-coding RNAs may have a cis-regulatory function. Here we show that, in addition to an unspliced form of Nepas, we can detect five alternatively spliced forms of Nespas up to 1.4 kb in length that are non-coding. The splice variants are paternally expressed; they start approximately 2 kb upstream of Gnasxl in a region of maternal methylation and end 2.5 kb beyond the ATG of Nesp. These variants do not correspond to exons of the human antisense transcript although they start in the same region; the Nespas transcript, like its human counterpart, is spliced in various alternative patterns. The identification of a set of small spliced imprinted transcripts in the human and now in the mouse suggests that these antisense transcripts are functionally important.

Transcriptional activation of alpha 1(III) procollagen gene in Tsk2/+ dermal fibroblasts.

Transient transfection experiments into Tsk2/+ and normal dermal fibroblasts were performed using four successively shorter Col3a1 promoter deletion constructs: #103, #110, #114, and #120 fused to the chloramphenicol-acetyl-transferase (CAT) reporter gene. The transcriptional activity in Tsk2/+ and normal dermal fibroblasts driven by the three longer constructs was equal. With the shortest construct, #120 (-96 to +16bp) the transcriptional activity in Tsk2/+ fibroblasts was 25 times higher than in normal fibroblasts. Electrophoretic mobility shift assays with a labeled #120 probe revealed that increased DNA-protein binding occurred with nuclear extracts prepared from Tsk2/+ fibroblasts and that this binding was displaced by consensus Sp1 and NF-1 oligonucleotide sequences. These data indicate that sequences from -96 to +16bp of the Col3a1 promoter play an important role in the upregulated expression of this gene in Tsk2/+ fibroblasts and that the promoter contains sequences which bind the trans-acting nuclear factors, Sp1(like) and NF-1(like).

A new mouse mutant, skijumper.

Low blood sugar levels are a well-known cause of severe illness and often death in newborn humans, especially those that are small for age. Few of the causes of neonatal hypoglycemia are known, and many remain to be found. We describe a novel mouse mutant, skijumper (skimp), in which pups, despite feeding well, have low levels of glucose and develop opisthotonos, followed by death typically within a few days after birth. Genetic mapping studies have localized the lesion to a approximately 1 cM interval on mouse Chromosome (Chr) 7 between D7Mit318 and D7Mit93. We have carried out extensive analysis to define the phenotype and its likely cause. In addition to low blood glucose, affected skijumper mice have lowglycogen and ketone levels. Mass spectrometric analysis of blood samples has excluded major defects in amino acid metabolism. Initial biochemical analyses suggested a defect in ketogenesis as one possible cause of this phenotype. However, measurements of levels and activities of carnitine, carnitine palmitoyl transferases, and other enzymes involved in ketogenesis, along with studies of mitochondrial structure and function, did not demonstrate significant differences between skijumper, unaffected littermates, and control wild-type mice. These results indicate that abnormal enzyme activity in known pathways does not appear to be the primary biochemical lesion in skijumper. The skijumper may be a new valuable model for studying and understanding one type of neonatal morbidity and death.

Novel phenotypes identified by plasma biochemical screening in the mouse.

We used ENU mutagenesis in the mouse for the rapid generation of novel mutant phenotypes for both gene function studies and use as new animal models of human disease (Nolan et al. 2000b). One focus of the program was the development of a blood biochemistry screen. At 8-12 weeks of age, approximately 300 ml of blood was collected from F1 offspring of ENU mutagenized male mice. This yielded approximately 125 ml of plasma, used to perform a profile of 17 standard biochemical tests on an Olympus analyzer. Cohorts of F1 mice were also aged and then retested to detect late onset phenotypes. In total, 1,961 F1s were screened. Outliers were identified by running means and standard deviations. Of 70 mice showing consistent abnormalities in plasma biochemistry, 29 were entered into inheritance testing. Of these, 9 phenotypes were confirmed as inherited, 10 found not to be inherited, and 10 are still being tested. Inherited mutant phenotypes include abnormal lipid profiles (low total and HDL cholesterol, high triglycerides); abnormalities in bone and liver metabolism (low ALP, high ALP, high ALT, and AST); abnormal plasma electrolyte levels (high sodium and chloride); as well as phenotypes of interest for the study of diabetes (high glucose). The gene loci bearing the mutations are currently being mapped and further characterized. Our results have validated our biochemical screen, which is applicable to other mutagenesis projects, and we have produced a new set of mutants with defined metabolic phenotypes.