Retroposon compensatory mechanism hypothesis not supported: Zfa knockout mice are fertile

It is hypothesized that autosomal retroposons compensate for the loss of their inactivated essential X-chromosome progenitors during spermatogenesis. Here we test this Retroposon Compensatory Mechanism (RCM) hypothesis using the Zfy gene family. The mouse autosomal retroposon Zfa is expressed in testes at the same developmental time points at which Zfx levels decline, which correspond to the time of male sex chromosome inactivation, suggesting that Zfa may compensate for the loss of Zfx during spermatogenesis. We examined the effect of Zfa-targeted mutagenesis on spermatogenesis in three genetically distinct mouse strains. Surprisingly, Zfa knockout mice showed no detectable fertility, sperm count, or testes morphology defects. We therefore conclude that Zfa is not an essential gene for spermatogenesis and fertility. This surprising finding now challenges the RCM hypothesis at least for the Zfy gene family. It also forces us to reevaluate the original data underpinning the RCM hypothesis for this family and to propose alternative hypotheses.     

Radiation-Resistant and Radiation-Sensitive Forms of Host Resistance to Polyomavirus

Newborn mice of several inbred strains develop few or no tumors following inoculation with highly tumorigenic strains of polyomavirus. Here we show that such resistant strains can be divided into two groups based on the responses of adult mice to radiation followed by virus inoculation. Most strains show a radiation-sensitive form of resistance (R^r-s) and develop tumors following radiation and virus challenge. This type of resistance has previously been recognized as immunological, based on T-cell responses against virus-encoded neoantigen(s) expressed in tumor cells. Other strains exhibit a radiation-resistant form of resistance (R^r-r) and fail to develop tumors when treated in the same manner. Three additional properties of R^r-r mice distinguish them from R^r-s mice: (i) survival of newborns following inoculation with a highly virulent and usually lethal strain of virus, (ii) resistance to virus spread in newborns inoculated with either tumorigenic or virulent virus strains, and (iii) dominant or semidominant transmission of resistance in crosses with a highly susceptible strain. The R^r-r phenotype reflects a constitutive nonimmunological type of resistance that is targeted to the virus and blocks its dissemination.     

Human sperm proteome: immunodominant sperm surface antigens identified with sera from infertile men and women.

The objective of this study was to identify those immunodominant sperm antigens recognized by antisperm antibodies (ASA) in the serum samples of infertile men and women. High-resolution two-dimensional gel electrophoresis was employed to separate human sperm proteins using isoelectric focusing or nonequilibrium pH gradient electrophoresis, followed by PAGE. Serum samples from 15 infertile male subjects and 6 infertile female subjects that contained ASA as assayed by the immunobead binding test (IBT) were analyzed by Western blotting followed by enhanced chemiluminescence (ECL). Serum samples from 10 fertile subjects (5 males and 5 females) that were ASA negative by IBT were used as controls. The ECL blots were analyzed by computer scanning to compare the immunoreactivity between serum samples from fertile and infertile subjects and to identify the antigens unique to the sera of the infertile subjects; 98 sperm auto- and iso-antigenic protein spots were recognized by sera from infertile males and females but not from fertile subjects. Based on vectorial labeling with 125I at the sperm surface, a subset of 6 auto- and iso-antigens was identified as possibly relevant to antibody-mediated infertility.     

Learning Rates and States from Biophysical Time Series: A Bayesian Approach to Model Selection and Single-Molecule FRET Data

Time series data provided by single-molecule Förster resonance energy transfer (smFRET) experiments offer the opportunity to infer not only model parameters describing molecular complexes, e.g., rate constants, but also information about the model itself, e.g., the number of conformational states. Resolving whether such states exist or how many of them exist requires a careful approach to the problem of model selection, here meaning discrimination among models with differing numbers of states. The most straightforward approach to model selection generalizes the common idea of maximum likelihood—selecting the most likely parameter values—to maximum evidence: selecting the most likely model. In either case, such an inference presents a tremendous computational challenge, which we here address by exploiting an approximation technique termed variational Bayesian expectation maximization. We demonstrate how this technique can be applied to temporal data such as smFRET time series; show superior statistical consistency relative to the maximum likelihood approach; compare its performance on smFRET data generated from experiments on the ribosome; and illustrate how model selection in such probabilistic or generative modeling can facilitate analysis of closely related temporal data currently prevalent in biophysics. Source code used in this analysis, including a graphical user interface, is available open source via http://vbFRET.sourceforge.net.     

Regulation of postsurgical fibrosis by the programmed death-1 inhibitory pathway

Surgical adhesions are a common and often severe complication of abdominal or pelvic injury that cause pelvic pain, bowel obstruction, and infertility in women. Current treatments are of limited effectiveness because little is known about the cellular and subcellular processes underlying adhesiogenesis. Recently, we showed that Th1 alpha beta CD4(+) T cells mediate the pathogenesis of adhesion formation in a rodent model of this disease process. In this study, we demonstrate that in mice these T cells home directly to the site of surgically induced adhesions and control local chemokine production in a manner dependent on the CD28 T cell costimulatory pathway. Conversely, the inhibitory programmed death-1 pathway plays a central role in limiting adhesiogenesis, as programmed death-1 blockade was associated with increased T cell infiltration, chemokine production, and a concomitant exacerbation of disease. Our results reveal for the first time that the development of postsurgical fibrosis is under the tight control of positive and negative T cell costimulation, and suggest that targeting these pathways may provide promising therapies for the prevention of adhesion formation.     

Expression cloning of an oxysterol 7alpha-hydroxylase selective for 24-hydroxycholesterol

The synthesis of 7alpha-hydroxylated bile acids from oxysterols requires an oxysterol 7alpha-hydroxylase encoded by the Cyp7b1 locus. As expected, mice deficient in this enzyme have elevated plasma and tissue levels of 25- and 27-hydroxycholesterol; however, levels of another major oxysterol, 24-hydroxycholesterol, are not increased in these mice, suggesting the presence of another oxysterol 7alpha-hydroxylase. Here, we describe the cloning and characterization of murine and human cDNAs and genes that encode a second oxysterol 7alpha-hydroxylase. The genes contain 12 exons and are located on chromosome 6 in the human (CYP39A1 locus) and in a syntenic position on chromosome 17 in the mouse (Cyp39a1 locus). CYP39A1 is a microsomal cytochrome P450 enzyme that has preference for 24-hydroxycholesterol and is expressed in the liver. The levels of hepatic CYP39A1 mRNA do not change in response to dietary cholesterol, bile acids, or a bile acid-binding resin, unlike those encoding other sterol 7alpha-hydroxylases. Hepatic CYP39A1 expression is sexually dimorphic (female > male), which is opposite that of CYP7B1 (male > female). We conclude that oxysterol 7alpha-hydroxylases with different substrate specificities exist in mice and humans and that sexually dimorphic expression patterns of these enzymes in the mouse may underlie differences in bile acid metabolism between the sexes.     

Gastric hyperplasia in mice lacking the putative Cdc42 effector IQGAP1

Human IQGAP1 is a widely expressed 190-kDa Cdc42-, Rac1-, and calmodulin-binding protein that interacts with F-actin in vivo and that can cross-link F-actin microfilaments in vitro. Recent results have implicated IQGAP1 as a component of pathways via which Cdc42 or Rac1 modulates cadherin-based cell adhesion (S. Kuroda et al., Science 281:832-835, 1998), whereas yeast IQGAP-related proteins have been found to play essential roles during cytokinesis. To identify critical in vivo functions of IQGAP1, we generated deficient mice by gene targeting. We demonstrate that IQGAP1 null mutants arise at normal frequency and show no obvious defects during development or for most of their adult life. Loss of IQGAP1 also does not affect tumor development or tumor progression, but mutant mice exhibit a significant (P < 0.0001) increase in late-onset gastric hyperplasia relative to wild-type animals of the same genetic background. While we cannot exclude that functional redundancy with IQGAP2 contributes to the lack of developmental phenotypes, the restricted expression pattern of IQGAP2 is not obviously altered in adult IQGAP1 mutant mice. Thus, IQGAP1 does not serve any essential nonredundant functions during murine development but may serve to maintain the integrity of the gastric mucosa in older animals.     

Further characterization of reproductive abnormalities in mCd59b knockout mice: a potential new function of mCd59 in male reproduction

CD59 is a GPI-linked membrane protein that inhibits formation of the membrane attack complex of complement. We reported recently that mice have two CD59 genes (termed mCd59a and mCd59b), and that the targeted deletion of mCd59b (mCd59b-/-) results in spontaneous hemolytic anemia and progressive loss of male fertility. Further studies of the reproductive abnormalities in mCd59b-/- mice reported in this study revealed the presence of abnormal multinucleated cells and increased apoptotic cells within the walls of the seminiferous tubules, and a decrease in the number, motility, and viability of sperm associated with a significant increase in abnormal sperm morphologies. Both the capacitation-associated tyrosine phosphorylation and the ionophore-induced acrosome reaction as well as luteinizing hormone, follicle-stimulating hormone, and testosterone serum levels were similar in mCd59b-/- and mCd59b+/+. Surprisingly, the functional deficiency of the complement protein C3 did not rescue the abnormal reproductive phenotype of mCd59b-/-, although it was efficient in rescuing their hemolytic anemia. These results indicate that the male reproductive abnormalities in mCd59b-/- are complement-independent, and that mCd59 may have a novel function in spermatogenesis that is most likely unrelated to its function as an inhibitor of membrane attack complex formation.