Genetic mapping of a male factor subfertility locus on mouse chromosome 4.

Male reproductive anomalies are widely distributed among mammals, and male factors are estimated to contribute to approximately 50% of cases of human infertility. The B10.M/Sgn (B10.M) mouse strain exhibits two adverse reproductive phenotypes: severe teratospermia and male subfertility. Although teratospermia is known to be heritable, the relationship between teratospermia and male subfertility has not been well characterized. The fertility of B10.M male mice is considerably lower (~ 30%) than that of standard laboratory mouse strains (~ 70%). To genetically analyze male subfertility, F2 males were produced by intercrossing the F1 progeny of female B10.M and male C3H/HeN mice. The fertility of each F2 male mouse was assessed based on the outcomes of matings with five females. Statistical analysis of correlations between the two reproductive phenotypes (teratospermia and subfertility) in F2 males (n = 177) revealed that teratospermia is not the cause of male subfertility. Quantitative trait loci (QTL) analysis of the male subfertility phenotype (n = 128) using GigaMUGA markers mapped one significant QTL peak to chromosome 4 at 62.9 centimorgans (cM) with a logarithm of odds score of 11.81 (P < 0.05). We named the QTL locus Mfsf1 (male factor subfertility 1). Further genetic analysis using recombinant males restricted the physical area to 1.53 megabasepairs (Mbp), encompassing 22 protein-coding genes. In addition, we found one significant QTL and one indicative QTL on chromosome 5 and 12, respectively, that interacted with the Mfsf1 locus. Our results demonstrate that genetic dissection of male subfertility in the B10.M strain is a useful model for characterizing the complex genetic mechanisms underlying reproduction and infertility.

A Cascade of epistatic interactions regulating teratozoospermia in mice.

Infertility in humans and subfertility in domestic animals are two major reproductive problems. Among human couples, ~15% are diagnosed as infertile, and males are considered responsible in about 50% of the cases. To examine male fertility, various sperm tests including analyses of sperm morphology, sperm count and sperm mobility are usually performed. Teratozoospermia, a condition characterized by the presence of morphologically abnormal sperm, is considered as a symptom of infertility. B10.MOL-TEN1 (TEN1) mice (Mus musculus) show inherited teratozoospermia at high frequencies (~50%). In this study, the polygenic control of teratozoospermia in the TEN1 strain was analysed. A quantitative trait loci analysis indicated three statistically significant loci, Sperm-head morphology 3 (Shm3; logarithm of the odds (LOD) score, 29.25), Shm4 (LOD score, 6.80), and Shm5 (LOD score, 3.58). These three QTL peaks were mapped to 24.3 centimorgans (cM) on chromosome 1, 32.0 cM on chromosome X, and 63.8 cM on chromosome 6, respectively. Another locus that is yet to be determined was also predicted. Shm3 was found to be the major locus responsible for teratozoospermia, and a sequential cascade of interactions of the other three loci was apparent. These results are expected to help understand the mechanisms underlying reproductive problems in humans or domestic animals.

Polygenic expression of teratozoospermia and normal fertility in B10.MOL-TEN1 mouse strain.

Subfertility and infertility are two major reproductive health problems in human and domestic animals. The contribution of the genotype to these conditions is poorly understood. To examine the genetic basis of male subfertility, we analyzed its relationship to sperm morphology in B10.MOL-TEN1 mice, which shows high-frequencies (about 50%) of morphologically abnormal sperm. Drastic histological changes were also found in the testis of the B10.MOL-TEN1. Segregation analysis showed that the abnormal sperm phenotype in B10.MOL-TEN1 was inherited and was predictably controlled by at least three loci. We also found that male fertility of this strain was normal. These findings indicate a complicated relationship between sperm morphology and male subfertility.

Spermatogenic defects in F2 mice between normal mouse strains C3H and C57BL/6 without mutation.

Genetic disorders are usually considered to be caused by harmful gene mutations, as well as by chromosomal aberrations, including small insertions, duplications and/or deletions. However, as infertile individuals often arise among the offspring of crosses between two fertile mouse strains, we postulate that a certain combination of 'normal' genes with neither gene mutations nor chromosomal aberrations can cause such serious phenotypic alterations as reproductive dysfunction. In this study, we show evidence that a combination of multiple normal genes from two different normal mouse strains manifests a wide range of male reproductive dysfunctions, from benign changes to complete infertility. These abnormal phenotypes are thought to have occurred by epistatic interactions of alleles.

QTL on mouse chromosomes 1 and 4 causing sperm-head morphological abnormality and male subfertility.

The B10.M mouse strain represents a model for male subfertility as it produces a significantly low number of offspring. The only known male reproductive phenotype of this strain is its high frequency of sperm-head morphological abnormalities (44.7 ± 2.4 %). We previously reported that this phenotype was the product of two recessive loci. In this study we mapped the loci causing the high frequency of sperm-head morphological abnormalities in this strain using F2 animals produced by crossing B10.M and C3H mice. Quantitative trait loci (QTL) analysis (n = 178) identified two recessive genes, one on Chromosome (Chr) 1 (LOD score = 30.585) and one on Chr 4 (LOD score = 4.532). Further analysis (n = 854) mapped the locus on Chr 1 between Ercc5 (23.55 cM) and D1Mit528 (25.95 cM) and the locus on Chr 4 between D4Mit148 (69.48 cM) and D4Mit170 (70.47 cM). It was also found that the effects of these two loci were not independent. The major locus on Chr 1 determines the expression of sperm-head abnormalities, while the locus on Chr 4 enhances the frequency of abnormalities only when the genotype of the Chr 1 locus is homozygous for the B10.M allele. The major locus on Chr 1 was named sperm-head morphology 1 (Shm1), while the modifier locus on Chr 4 was named sperm-head morphology 2 (Shm2).

Inherited sperm head abnormalities in the B10.M mouse strain.

Examination of sperm head morphology is one of the requisite tests of the functional capacity of semen in reproduction. In the present study, genetic effects on morphological sperm head abnormalities in mice were investigated. The frequency of abnormal spermatozoa was determined in 17 inbred mouse strains and it was found that strain B10.M had the highest frequency of abnormal spermatozoa (44.7%). Segregation analysis was then used to show that the abnormal sperm phenotype in B10.M mice was inherited. The results indicated that this sperm abnormality was controlled by two distinct recessive alleles. It is proposed that the high frequency of the heritable abnormal sperm phenotype in the mouse B10.M strain explains the subfertility of this strain, as evidenced by its reduced litter size.

The origin and genetic variation of domestic chickens with special reference to junglefowls Gallus g. gallus and G. varius.

It is postulated that chickens (Gallus gallus domesticus) became domesticated from wild junglefowls in Southeast Asia nearly 10,000 years ago. Based on 19 individual samples covering various chicken breeds, red junglefowl (G. g. gallus), and green junglefowl (G. varius), we address the origin of domestic chickens, the relative roles of ancestral polymorphisms and introgression, and the effects of artificial selection on the domestic chicken genome. DNA sequences from 30 introns at 25 nuclear loci are determined for both diploid chromosomes from a majority of samples. The phylogenetic analysis shows that the DNA sequences of chickens, red and green junglefowls formed reciprocally monophyletic clusters. The Markov chain Monte Carlo simulation further reveals that domestic chickens diverged from red junglefowl 58,000+/-16,000 years ago, well before the archeological dating of domestication, and that their common ancestor in turn diverged from green junglefowl 3.6 million years ago. Several shared haplotypes nonetheless found between green junglefowl and chickens are attributed to recent unidirectional introgression of chickens into green junglefowl. Shared haplotypes are more frequently found between red junglefowl and chickens, which are attributed to both introgression and ancestral polymorphisms. Within each chicken breed, there is an excess of homozygosity, but there is no significant reduction in the nucleotide diversity. Phenotypic modifications of chicken breeds as a result of artificial selection appear to stem from ancestral polymorphisms at a limited number of genetic loci.

Unique inbred strain MSM/Ms established from the Japanese wild mouse.

Most laboratory mice belong to a species of house mouse, Mus musculus. So far, at least three subspecies groups have been recognized; domesticus subspecies group (DOM) distributed in western Europe, musculus subspecies group (MUS) distributed in eastern Europe and northeast Asia, and castaneus subspecies group (CAS) found in southwest and southeast Asia including southern China. These subspecies are estimated to have branched off roughly one million years ago. Genetic comparison between subspecies' groups and common inbred strains (CIS) have revealed that the genetic background of CIS is derived mainly from DOM. This shows the importance of non-DOM wild mice as valuable genetic resources. We started to establish our unique strain, MSM/Ms, from MUS in Japan in 1978. In the beginning, we kept wild mice trapped in Mishima in large plastic buckets. In 1979, breeding by sister-brother mating started. The MSM/Ms inbred strain was established in 1986 and 21 years later it reached F(100). During breeding, no significant fluctuations in litter size and sex ratios have been observed. Extensive genetic analyses of chromosome C-banding pattern, biochemical markers and microsatellite DNA (MIT) markers of this strain have demonstrated the characteristics of MUS. A phylogenetic tree constructed from MIT markers has confirmed the MUS nature of MSM strain. Taken together with its genetic remoteness from CIS, MSM appears to maintain many valuable alleles for investigation of biological functions and diseases. Some of these alleles have avoided selection during breeding as either fancy mice or laboratory mice. The MSM-specific genetic traits discovered to date are discussed.

Pseudomonas aeruginosa, under DNA replication inhibition, tends to form biofilms via Arr.

Bacteria infecting eukaryotic hosts often encounter therapeutic antimicrobial and DNA damaging agents and respond by forming biofilms. While mechanisms of biofilm response are incompletely understood, they seem to involve bacterial second messenger bis-(3'-5')-cyclic dimeric guanosine monophosphate (c-di-GMP) signaling. We hypothesized that DNA replication inhibition induces bacterial biofilm formation via c-di-GMP signaling. Evidently, we found that Pseudomonas aeruginosa mounted a biofilm response to the subinhibitory DNA replication inhibitors hydroxyurea and nalidixic acid, but planktonic proliferation was inhibited. The biofilm response was suppressed either genetically by mutations causing planktonic resistance or biochemically by reversal of replication inhibition. Biofilms were induced by a mechanism of stimulated adhesion of planktonic filaments having impaired DNA replication, as examined under fluorescence microscopy. Induction was suppressed by either inhibition or mutation of Arr-a c-di-GMP phosphodiesterase. These results suggest that P. aeruginosa, under DNA replication stress, tends to form biofilms via Arr. The profound implications of the SOS response, planktonic-sessile and bacteria-cancer relationships are discussed.

Management of prenatal ovarian cysts.

OBJECTIVES: The aim of the present study was to analyze the antenatal and postnatal outcome of fetal ovarian cysts in relation to their ultrasonographic pattern and size. METHODS: Sixteen fetal ovarian cysts were diagnosed in 16 fetuses and followed with serial ultrasonograms in utero and after birth until spontaneous or surgical resolution. RESULTS: Eleven fetal ovarian cysts were simple cysts at first prenatal scan but 3 of the 11 became complex cysts at last prenatal scan and required postnatal laparoscopic surgery. Seven of the 11 simple cysts (63%) disappeared on follow-up imaging by ultrasonograms or MRI during pregnancy or within 2 months after birth. The rate of spontaneous resolution of simple cysts was higher than that of complex cysts (40.0%). The mean maximum diameter of the ovarian cysts before delivery that were subsequently excised surgically at postnatal period (50+/-13.4 mm) was not different from that of ovarian cysts that resolved spontaneously (42.8+/-12.8 mm, P=0.2918). CONCLUSION: In our study, cyst size did not predict the risk of ovarian loss. The opportunity of laparoscopic exploration versus conservative management needs to be investigated because some complex cysts resolved spontaneously in the postnatal period.

Cell-surface streptavidin fusion protein for rapid selection of transfected mammalian cells.

We developed a series of eight mammalian cell surface marker fusion genes by using the streptavidin gene from Streptomyces avidinii. These fusion genes are useful and non-growth-toxic selection markers for rapid-harvest transfected mammalian cells. Two streptavidin constructs were used; the longer fragment contains the native bacterial signal sequence, which the shorter fragment lacks. For expression of the streptavidin antigen on the surface of mammalian cells, streptavidin was flanked by a mammalian signal sequence and a transmembrane domain (from mouse H2-K or Kit); some constructs also contained the gene for enhanced green fluorescent protein (EGFP). We transfected a series of plasmids encoding the fusion proteins into HeLa cells and determined that the transfected cells produced the fusion protein on their cell surfaces. To separate transfected cells from nontransfected cells, we incubated cells with a polyclonal antibody against streptavidin, and antibody-bound cells were harvested by the use of paramagnetic beads coupled with the corresponding secondary antibody. We obtained highly pure populations of transfected cells; this result also confirmed the production of the fusion protein on the cell surface. Cell growth assays revealed that none of the transfected fusion genes or their products adversely affected the proliferation of HeLa cells. Our results indicate that the fusion constructs we developed and the immunomagnetic separation protocol we used are valuable tools for various transfection applications. In particular, the constructs containing EGFP are advantageous because transfection efficiency can be assessed without additional treatment of cells.

Anti-bovine CD34 monoclonal antibody reveals polymorphisms within coding region of the CD34 gene.

OBJECTIVE: Monoclonal antibodies (mAbs) against CD34 are widely used for purification of CD34+ hematopoietic as well as nonhematopoietic stem/progenitor cells. We produced mAbs against bovine CD34 (boCD34) to facilitate the study of hematopoiesis in cattle. METHODS: MAbs were produced by immunizing BALB/c mice with BALB/3T3 cells transfected with boCD34 cDNA. Staining of bone marrow mononuclear cells (BMMNCs) from 10 newborn Holstein calves with the mAbs was examined by flow cytometry. The nucleotide sequence of the coding region for boCD34 in each calf was determined after amplification of the cDNA by reverse-transcription polymerase chain reaction (RT-PCR). BoCD34 fusion proteins, each representing one of the boCD34 alleles found to exist in the calves, were expressed in HeLa cells by DNA transfection, and the staining of these proteins with the mAbs was assessed. RESULTS: One mAb, N21, stained relatively high percentages of BMMNCs from 4 calves but failed to stain those from the other calves. RT-PCR analysis revealed single-nucleotide polymorphisms within the coding region, 3 of which led to amino-acid substitutions. A CD34 mutation experiment indicated that mAb N21 bound to a boCD34 allele with tryptophan at amino acid 167 but not to that with arginine. CONCLUSION: By using mAb N21 as an allelic cell marker, it would be feasible to detect and isolate boCD34+ cell species derived from N21+ donors in N21- recipients following allogeneic in utero transplantation; this would make cattle potentially useful as large animal models with a unique experimental advantage.

Dynamic rearrangement of telomeres during spermatogenesis in mice.

Chromosomal structure within the nucleus influences various biological processes such as transcription and replication. Telomeres are located at the end of eukaryotic chromosomes and they can be a decisive factor for correct chromosomal positioning. To gain new insight into telomere dynamics, we examined telomere length and positional changes during spermatogenesis using improved fluorescence in situ hybridization (FISH) and in situ telomeric repeat amplification protocols (TRAP) on histological sections. FISH revealed telomere length and chromosome position within nuclei change dynamically. Telomere extension occurred during spermiogenesis. In situ TRAP analysis verified elevated telomerase activity in elongating spermatids. Together, these data show that elongated spermatids have longer telomeres than precursor spermatogenic cells. This observation indicates that telomere elongation in haploid cells occurs after meiosis and in the absence of genomic replication. Analyses of testes from telomerase null mice further support the significance of telomere dynamics during spermatogenesis and the existence of an alternative telomere extension pathway.

General anesthesia of infant mice by isoflurane inhalation for medium-duration surgery.

In this report we describe a means of general anesthesia for medium-duration (i.e., 20 to 60 min) surgery of infant mice. We tested isoflurane inhalation (2.0% isoflurane in air or oxygen during induction, and 1.5% after surgical anesthesia) anesthesia of 6-to 10-day-old C57BL/6JJcl mice and obtained safe, effective, and reproducible results.

Intracellular expression of the Salmonella plasmid virulence protein, SpvB, causes apoptotic cell death in eukaryotic cells.

The spv genes carried on the Salmonella virulence plasmid are commonly associated with severe systemic infection in experimental animals. The SpvB virulence-associated protein has been shown to ADP-ribosylate actin, and this enzymatic activity is essential for virulence in mice. Here, we present evidence that intracellular expression of SpvB protein induces not only disruption of actin filaments but also apoptotic cell death in eukaryotic cells.

Extracellular secretion of the virulence plasmid-encoded ADP-ribosyltransferase SpvB in Salmonella.

Nontyphoid Salmonella enterica requires the plasmid-encoded spv genes to establish successful systemic infection in experimental animals. The SpvB virulence-associated protein has recently been shown to contain the ADP-ribosyltransferase domain. SpvB ADP-ribosilates actin and depolymerizes actin filaments when expressed in cultured epithelial cells. However, spontaneous secretion or release of SpvB has not been observed under in vitro growth conditions. In the present study we investigated the secretion of SpvB from Salmonella using in vitro and in vivo assay systems. We showed that SpvB is secreted into supernatant from Salmonella strains that contain the cloned spvB gene on a plasmid when they grew in intracellular salts medium (ISM), a minimal medium mimicing the intracellular iron concentrations of eukaryotic cells. A series of mutant SpvB proteins revealed that an N-terminal region of SpvB located at amino acids 1-229 was sufficient to promote secretion into extracellular milieu. Confocal immunofluorescence microscopy also demonstrated efficient localization of the N-terminal domain of SpvB(1-360) tagged with biotinylated peptide within infected host cell cytosol but not truncated SpvB(1-179) fusion protein. In addition, mutations that inactivate genes within Salmonella pathogenicity island 1 or Salmonella pathogenicity island 2 that encode type III secretion systems (TTSS) could secrete the SpvB protein into the culture medium. These results indicate that SpvB protein is transported from the bacteria and into the host cytoplasm independent of TTSS.