Mapping of an immotile short tail sperm defect in the Finnish Yorkshire on porcine Chromosome 16

An immotile short tail sperm defect has recently been identified as a hereditary disorder present within the Finnish Yorkshire pig population. The syndrome is inherited as an autosomal recessive disease exclusively expressed in male individuals as shorter sperm tail length and immotile spermatozoa. Based on the assumption of a recent common origin of the disease-causing mutation, a genome-wide search was performed with 228 evenly spaced microsatellites by homozygosity mapping of affected and unaffected DNA pools. One locus, SW2411 on Chr 16, demonstrated a significantly skewed allele distribution between the two pools. Linkage analysis of five markers in this region mapped the disease-causing gene within a 6-cM confidence interval region with a highest LOD score of 7.7 at marker SW419. It appears that three-marker haplotypes can be used for marker-assisted selection within analyzed pedigrees. Furthermore, future fine mapping may reveal a more precise population-wide associated haplotype and facilitate identification of a new gene affecting sperm tail development. Received: 5 July 2001 / Accepted: 13 September 2001     

The Dag defect of the tail of the bull sperm. Studies on the inheritance and pathogenesis

During the period 1963–1979, 15 spontaneous cases of the sterilizing Dag defect of the sperm tail in Danish Jersey bulls have been examined. The pedigree of these bulls could be traced back through both paternal and maternal lines to a common ancestor bull born in 1934. The probable heredity of the defect was tested in a sire-daughter breeding experiment. Six bulls out of 38 sons born produced semen typical of the Dag defect thus confirming that the defect is due to the presence of an autosomal recessive factor. Systematic examination of the epididymal contents from 17 bulls revealed that the defect consistently developed in the distal part of the epididymal caput. Neither biophysical and biochemical qualities of the epididymal contents nor the histological appearance of the duct epithelium differed from the findings in normal bulls.     

Molecular cloning and characterization of oppo 1: a haploid germ cell-specific complementary DNA encoding sperm tail protein

We isolated a cDNA clone specifically expressed during spermatogenesis from a subtracted cDNA library of mouse testis. The cDNA consisted of 1085 nucleotides and had an open reading frame of 870 nucleotides encoding a putative protein of 290 amino acid residues. Northern blot analysis revealed a 1.2-kilobase mRNA exclusively expressed in the testis in adult mice; the mRNA was first detected late pachytene stage, and expression increased as the animals matured. The protein encoded by the mRNA had a molecular weight of approximately 33 kDa by Western blot analysis, and was localized to occupy the flagella from the connecting piece through the principal piece. We named this newly isolated gene oppo 1, and we suggest that it plays an important role in sperm tail structure and/or sperm movement.     

Molecular cloning of an acrosomal sperm antigen gene and the production of its recombinant protein for immunocontraceptive vaccine

A monoclonal antibody, HS-63, which reacts specifically with a highly conserved sperm acrosome antigen, was shown to inhibit in vitro fertilization of mouse and human. The corresponding sperm antigen designated as MSA-63 was purified to homogeneity from mouse testes and used as an immunogen to generate polyclonal antisera in rabbits. The cDNA fragments of MSA-63 gene were cloned from mouse testis cDNA library by an immunoscreening method using polyclonal antisera specific for MSA-63. Using the established cDNA clone as a probe, the gene encoding for MSA-63 protein was found to be conserved among different mammalian species. Only one specific mRNA 1.5 kb in size was identified from the adult mouse testis among different mouse tissues. The recombinant fusion protein containing MSA-63 protein fragment was produced in Escherichia coli and used to immunize female mice. Similar to the original HS-63 monoclonal antibody, the antisera thus produced reacted only with the sperm acrosome and revealed significant inhibition to the in vitro fertilization of mouse oocytes. The results of this preliminary study suggest that it is feasible to mass produce sperm-specific antigens or their antigenic fragments by recombinant DNA technology for the development of sperm antigen-based immunocontraceptive vaccines.     

Alu-PCR: characterization of a chromosome 6-specific hybrid mapping panel and cloning of chromosome-specific markers.

The Alu-polymerase chain reaction (Alu-PCR) was applied to selectively amplify DNA sequences from human chromosome 6 using a single primer (A1) directed to the human Alu consensus sequence. A specific amplification pattern was demonstrated for a panel of eight somatic cell hybrids containing different portions of chromosome 6. This PCR pattern permits the identification of submicroscopic DNA alterations and can be utilized as a reference for additional chromosome 6-specific hybrids. To obtain new chromosome 6-specific markers we established two libraries from PCR-amplified sequences using two somatic cell hybrids (MCH381.2D and 640-5A). Out of a total of 109 clones that were found to be chromosome 6 specific, 13 clones were regionally assigned. We also included a procedure that allows the isolation of chromosome 6-specific markers from hybrids that contain human chromosomes other than 6. Our results will contribute to the molecular characterization of chromosome 6 by fostering characterization of somatic cell hybrids and by the generation of new regionally assigned DNA markers.     

Human 6-pyruvoyltetrahydropterin synthase: cDNA cloning and heterologous expression of the recombinant enzyme.

6-Pyruvoyl-tetrahydropterin synthase (PTPS) is involved in the biosynthesis of tetrahydrobiopterin (BH4), an essential cofactor for enzymes such as the hepatic phenylalanine hydroxylase. BH4 deficiency causes malignant hyperphenylalaninemia. We cloned the human liver cDNA encoding PTPS. The coding region for PTPS contains 145 amino acids and predicts a polypeptide of 16'387 Da. The human amino acid sequence showed a 82% identity with the rat liver sequence. Expression of the cDNA in E. coli yielded the active enzyme and showed immunoreactivity with antibodies against the rat liver PTPS. This is the basis for the molecular understanding of BH4 deficiency in patients suffering from a defect in PTPS activity.     

Molecular assembly and structure of the bacteriophage T4 tail

The tail of bacteriophage T4 undergoes large structural changes upon infection while delivering the phage genome into the host cell. The baseplate is located at the distal end of the contractile tail and plays a central role in transmitting the signal to the tail sheath that the tailfibers have been adsorbed by a host bacterium. This then triggers the sheath contraction. In order to understand the mechanism of assembly and conformational changes of the baseplate upon infection, we have determined the structure of an in vitro assembled baseplate through the three-dimensional reconstruction of cryo-electron microscopy images to a resolution of 3.8 Å from electron micrographs. The atomic structure was fitted to the baseplate structure before and after sheath contraction in order to elucidate the conformational changes that occur after bacteriophage T4 has attached itself to a cell surface. The structure was also used to investigate the protease digestion of the assembly intermediates and the mutation sites of the tail genes, resulting in a number of phenotypes.     

Mapping of three translocation breakpoints associated with orofacial clefting within 6p24 and identification of new transcripts within the region

Orofacial clefting (OFC) is a common congenital malformation. Here we report the refinement of three translocation breakpoints of patients exhibiting OFC within the 6p24 region, and the isolation and characterisation of novel genes, one of which is directly disrupted by the translocation breakpoint of a patient. The gene has been characterized and orthologues identified in bovine, murine and pufferfish.     

ICSI精子遗传和表观遗传学缺陷

卵胞浆内精子注射(Intracytoplasmic sperm injection, ICSI)技术可用于男性少精、弱精、精子畸形、无精子和常规体外受精周期失败等, 克服了精子数量不足甚至直接从附睾、睾丸获取精子来治疗不育。该技术直接将单个精子注射入卵子, 因违背自然受精的生物学法则而具有很大的遗传风险。文章对ICSI精子遗传缺陷和表观遗传缺陷及其相关疾病进行综述, 可进一步认识ICSI精子遗传与表观遗传缺陷导致后代遗传风险增加的分子的机理, 文章阐述了ICSI精子有待于通过DNA甲基化、组蛋白乙酰化等表观遗传因子进行严格质量控制, 切实降低ICSI遗传及表观遗传缺陷风险的必要性。     

Molecular cloning and recombinant expression of cytochrome P450 CYP6B6 from Helicoverpa armigera in Escherichia coli

The cytochrome P450 s play a significant role in the detoxification of plant allelochemicals and synthetic insecticides in Lepidoptera. In the cotton bollworm Helicoverpa armigera, 2-tridecanone and quercetin can induce P450-dependent monooxygenase activity increased, to further the characterization of P450, the CYP6B6 of cotton bollworm (H. armigera) was cloned, sequenced and expressed in pMAL-p2x vector and expressed in Escherichia coli. The deduced amino acid sequences of cytochrome P450 in the midgut and fat body of H. armigera showed 98.23 and 97.84 % similarity with CYP6B6, respectively. According to nomenclature of P450 s, the P450 genes we got belong to CYP6B. Purification of recombinant protein based on the affinity of MBP for maltose was achieved by Mal-Tag magnetic beads. The purified protein was used to raise polyclonal antibody according to classical procedure. SDS–PAGE and Western blot results indicated that MBP-CYP6B6 had been successfully expressed. The ethoxycoumarin-O-deethylase activity of the purified recombinant protein was 36.5 ± 8.12 pmol of 7-hydroxycoumarin/min/mg protein, which showed the fusion MBP-CYP6B6 had the ability to o-deethylase of 7-ethoxycoumarin.     

An axonemal dynein at the Hybrid Sterility 6 locus: implications for t haplotype-specific male sterility and the evolution of species barriers

Poor sperm motility characterized by a distinct aberration in flagellar waveform known as ``curlicue' is a hallmark of t haplotype (t) homozygous male sterility. Previous studies have localized ``curlicue' and a flagellar developmental defect, ``whipless', to the Hybrid Sterility 6 locus (Hst6), between the markers Pim1 and Crya1. More recent heterospecific breeding experiments between Mus spretus (Spretus) and Mus musculus domesticus (Domesticus) have mapped the primary source(s) of both ``curlicue' and ``whipless' to a small sub-locus of Hst6, Curlicue a (Ccua). Here we report the complete physical isolation of the Ccua locus and the identification of a candidate gene for expression of both ``whipless' and ``curlicue' at its proximal end, an axonemal dynein heavy chain gene, Dnahc8, formerly mapped by interspecific backcross analysis near Pim1. Dnahc8 mRNA expression commences in the Domesticus wild-type testis just prior to flagellar assembly and is testis-specific in the adult male. However, expression of Dnahc8 is not readily evident in the testis of either Spretus or ``whipless' animals (Domesticus males homozygous for the Spretus allele of Dnahc8). Our results argue that Dnahc8 is fundamental to flagellar organization and function in Domesticus, but not Spretus, and suggest that Dnahc8 is integral to both Hst6- and t-specific male infertility. Received: 13 July 1999 / Accepted: 25 August 1999     

Sterility and Hypermutability in the P-M System of Hybrid Dysgenesis in DROSOPHILA MELANOGASTER

Inbred wild strains of Drosophila melanogaster derived from the central and eastern United States were used to make dysgenic hybrids in the P-M system. These strains possessed P elements and the P cytotype, the condition that represses P element transposition. Their hybrids were studied for the mutability of the P element insertion mutation, snw, and for the incidence of gonadal dysgenesis (GD) sterility. All the strains tested were able to induce hybrid dysgenesis by one or both of these assays; however, high levels of dysgenesis were rare. Sets of X chromosomes and autosomes from the inbred wild strains were more effective at inducing GD sterility than were sets of Y chromosomes and autosomes. In two separate analyses, GD sterility was positively correlated with snw mutability, suggesting a linear relationship. However, one strain appeared to induce too much GD sterility for its level of snw destabilization, indicating an uncoupling of these two manifestations of hybrid dysgenesis.     

Mutations in a novel locus on mouse chromosome 11 resulting in male infertility associated with defects in microtubule assembly and sperm tail function

Traditional gene knock-out approaches using homologous recombination in embryonic stem cells are routinely used to provide functional information about genes involved in reproduction. In the present study, we examined a novel approach using N-ethyl-N-nitrosourea (ENU) together with a balancer chromosome mating strategy to identify new loci with functional roles in male fertility. Our genetic strategy is a forward-genetic approach; thus, our phenotypic investigation begins with the discovery of an abnormal phenotype without previous knowledge of the mutant locus. We isolated eight recessive mutations on chromosome 11 that resulted in male or female infertility from a screen of 184 founder pedigrees from ENU-treated males. After testing the six male infertile and two female infertile mutations for their ability to complement, we found that three independent recessive male infertile mutations failed to complement each other. The male infertility was associated with reduced epididymal sperm count, a block in late-spermatid differentiation, and increased apoptosis. Furthermore, the three male infertile mutants had severe defects in epididymal sperm morphology associated with incorrect microtubule assembly. Electron microscopy revealed unique defects in sperm head and tail morphology for each of the three alleles. One allele had an abnormal manchette assembly of the sperm head. The other two alleles had different abnormalities in the 9+2 patterning of the microtubules in the sperm tail axoneme, with one containing only five of the microtubule doublets and the other containing an extra doublet. The isolation of this allelic series identifies a new locus on mouse chromosome 11 that is required for spermiogenesis and male fertility.     

Morphogenesis of the decapitated and decaudated sperm defect in two brothers

The “decapitated sperm” defect, found in both of two sterile brothers, may be assumed to have a genetic origin. The present material suggests that the term “decapitated spermatozoa” is not exact, because detached heads and tails were found in the brothers' ejaculate that could be regarded as “decapitated tails” and “decaudated heads.” The present report describes frequent, more or less advanced stages of detachment. Both heads and tails showed a normal structure in which only the postnuclear region was deficient, lacking basal plate and implantation fossa. A break at a different level of the midpiece, and therefore three kinds of separation, were observed. The defect, according to the present research, must originate in the testicular region, whereas the detachment occurs in the epididymis.     

Mapping and cloning of the par-region of broad-host-range plasmid RP4

Various deletion mutants of the identical broad-host-range plasmids RP4 and RK2, obtained after conjugative transfer of these plasmids from Escherichia coli to Alcaligenes eutrophus H16, were tested with respect to their segregation behaviour. Although the parent plasmids and some of the deletion mutants were completely stable in both A. eutrophus and E. coli, other derivatives were lost under non-selective conditions. The analysis of these deletion mutants allowed the identification and mapping of a region encoding a partitioning system (par) between the tra2 region and the kanamycin resistance gene of RP4 (RK2). This area corresponds to the PstI-C restriction fragment of RP4 (RK2). Cloning of this fragment into several unstable vector plasmids including pBR322 and pACYC177 resulted in all cases in an increase of segregational stability. By insertion of the par-region into an unstable broad-host-range mobilizable plasmid and transfer to a series of gram-negative bacteria, it could be shown that the cloned par-region of RP4 is functional in a broad-host-range.     

Connecting bridges between axoneme and other components in the sperm tail of some insects

Spermatozoa from three insect groups were examined by electron microscopy and found to have bridges that connect some of the axonemal doublets with either the two mitochondrial derivatives or, in the phasmids, the so-called laminated bodies. Within Hemiptera Heteroptera the bridges extend from doublet Nos. 1 and 5, within chrysopid neuropterans from doublet Nos. 2 and 5, and in the phasmids from axonemal doublet Nos. 2 and 4. Bridges were looked for in spermatozoa from several other insect groups but not found. The bridges in the chrysopids are regularly curved rather than straight. While bridges in heteropterans and chrysopids were seen in spermatozoa fixed with “standard fixatives,” those in the phasmids were distinctly resolved only in spermatozoa that had been fixed with a tannic acidcontaining fixative. In spite of these differences, it is conceivable that the bridges in these three insect taxa are all derived from similar, faint, bridge-like connections that sometimes can be seen to extend from all or many doublets toward the axonemal sheath of the early insect spermatid. These bridges or bridge-like structures might have a morphogenic function in that they may specify the location of the mitochondria later to become mitochondrial derivatives or, in the phasmids, of the laminated bodies.