Beneficial effects of microsurgical varicocoelectomy on sperm maturation, DNA fragmentation, and nuclear sulfhydryl groups: a prospective trial.

There is evidence to show that varicocele repair can improve conventional sperm parameters but the effects on sperm chromatin integrity have not been fully elucidated. We sought to examine the effects of varicocelectomy on sperm maturation, nuclear chromatin integrity and nuclear sulfhydryl groups. We conducted a prospective study of consecutive infertile men (n=29) that underwent a microsurgical sub-inguinal varicocelectomy for treatment of a clinically palpable varicocele and abnormal semen parameters. Six healthy sperm donors served as controls. We evaluated conventional sperm parameters and markers of sperm chromatin and DNA integrity (aniline blue (AB) staining, iodoacetamide fluorescein (IAF) fluorescence and, % DNA fragmentation index (%DFI) and percent high DNA stainability (%HDS) by sperm chromatin structure assay) before and 6 months after surgery. The sperm %DFI, %HDS, % 5-IAF staining (diffuse head staining) and % AB staining (dark blue) were all significantly lower in the control group compared to infertile men with varicocele (8 vs. 20%, 4.0 vs. 9.6%, 1.7 vs. 16.3%, and 2.5 vs. 13.5% respectively). The %HDS and %DFI decreased significantly after surgery (from 10% to 6% and from 20% to 13%, respectively). Similarly, the %5-IAF and %AB staining also decreased significantly after surgery (from 16.3% to 5.4%, and from 13.5% to 5.4%, respectively). We observed significant inversely relationships between sperm progressive motility and both %IAF staining and %DFI (r=-0.44 and -0.43, respectively). The data show that varicocelectomy is associated with an improvement in sperm DNA integrity and chromatin compaction using three different assays of sperm chromatin integrity.

Human sperm chromatin undergoes physiological remodeling during in vitro capacitation and acrosome reaction.

Capacitation (CAP) and acrosome reaction (AR) are sequential processes of sperm activation. Beside the known ionic, membrane, and transduction events and final release of proteolytic enzymes that help sperm movement toward the egg, chromatin changes, such as a physiological remodeling, are also possible. Our aims were to ascertain that CAP and AR do not induce DNA damage and to evaluate changes occurring in the human sperm head during these physiological processes using cytochemical stains. Percollpurified spermatozoa from normal donors were incubated in Biggers, Whitten, and Whittingham medium ± fetal cord serum ultrafiltrate (CAP inducer) and then with lysophosphatidylcholine (AR inducer). CAP and AR were associated with increases in aniline blue (AB, for histones; ∼70%) and toluidine blue (TB, for chromatin compaction; ∼40%) staining but had no influence on that of chromomycin A3 (for protamines). The increase (∼40%) in iodoacetamide-fluorescein (IAF, for sulfhydryl groups) staining observed during CAP was absent after AR. CAP and AR did not damage DNA (percentage of DNA fragmentation index remained low) nor affect histone content. CAP, and even more AR, primed sperm heads to decondense (∼80% and ∼140% increases, respectively) when challenged with sodium dodecyl sulfate + dithiothreitol. Interestingly, induced decondensation correlated with all other tests (CAP, AB, TB, and IAF). Therefore, the data strongly support a physiological remodeling of nondamaged human sperm chromatin during CAP and AR, and modifications are probably interlinked and help prepare chromatin for postfertilization events.

Identification of canine calicivirus capsid protein and its immunoreactivity in western blotting.

A canine calicivirus (CaCV) isolated in Japan, designated as CaCV No. 48 strain, was propagated in MDCK cells and purified by CsCl equilibrium gradient centrifugation. Sodium dodecyl sulfate-polyacrylamide gel electrophoretic analysis of the purified samples revealed the presence of only one major species of viral protein of about 60 kilodaltons after Coomassie staining. The same band, presumably that of the capsid protein, was detected by western blotting using a mouse hyperimmune serum. This capsid protein was synthesized in MDCK cells as early as 2 hr post-inoculation. Experimental infection of dogs resulted in the production of anti-CaCV antibodies which were detected by microneutralization test and western blotting. Likewise, serosurvey revealed not only the presence of neutralizing antibodies but also reactivity of the field sera against the capsid protein of the purified virus. These results indicate that the capsid protein of CaCV No. 48 strain is immunogenic and could be detected by antibodies in western blotting.

Isolation of canine parvovirus from a cat manifesting clinical signs of feline panleukopenia.

Twenty-seven feline parvovirus (FPV) isolates were recovered from cats clinically diagnosed with feline panleukopenia (FPL) for assessing antigenic and genomic properties of FPL viruses (FPLV) recently prevalent among cats in Japan. All isolates, with the exception of one novel isolate, FPV-314, possessed homologous properties, and their subgroups in FPVs were identified as FPLV. The FPV-314 isolate, which was from a 1.5-year-old cat which manifested clinical signs of FPL and died on the 13th day after the first medical examination, was finally identified as canine parvovirus (CPV) because it lacked a specific antigenic epitope commonly detected in FPLV and mink enteritis virus and because the nucleotide sequence of the capsid protein gene was almost identical to those of CPV-2a and -2b antigenic type strains recently prevalent among dogs in Japan. The present result together with our previous findings (M. Mochizuki, R. Harasawa, and H. Nakatani. Vet. Microbiol. 38:1-10, 1993) indicates the possibility that CPV and FPLV undergo mutual interspecies transmission between dogs and cats, and it is postulated that they may cause disease in some adventitious hosts.

Expression in E. coli and purification of the active autoprotease P20 of classical swine fever virus.

The genes for Flaviviridae structural proteins are located at the 5' terminus of the genome, while the 3' terminus contains the genes for the non-structural proteins. The first protein product of the large ORF of pestiviruses, the p20 protein, is however a non-structural protein which possess an autoproteolytic activity. Here we report the cloning of the p20/p14 genes behind the strong Trc promotor and expression of the p20 at high levels in E. coli. The autoprotease p20 was responsible for its own release from the nascent polyprotein in E. coli and was further purified by chromatography techniques.

Comparison of polymerase chain reaction with virus isolation and haemagglutination assays for the detection of canine parvoviruses in faecal specimens.

A polymerase chain reaction (PCR) assay, which specifically amplifies the capsid gene of canine parvovirus (CPV), was compared as a diagnostic method for detecting CPV in faeces, with virus isolation (VI) on Crandell feline kidney (CRFK) or Madin-Darby canine kidney (MDCK) cells, and a faecal haemagglutination (HA) assay confirmed by inhibition with a CPV-specific antiserum. Although a false-negative result was obtained in one of 59 faecal samples (1.7 per cent) tested by the PCR assay, it was as sensitive as the VI assay using MDCK cells, and more sensitive than the VI assay using CRFK cells or the HA assay. These results indicate that the PCR assay may be useful as a routine diagnostic method for detecting CPV in faecal specimens.

Polymerase chain reaction-mediated cloning and in vitro translation of the genes coding for the structural proteins of hog cholera virus.

After amplification by PCR, the 5' region of the genome of hog cholera virus (HCV) strain Alfort 187 was cloned and sequenced. The nucleotide and deduced amino acid sequences were compared with the ones of other pestiviruses. By in vitro translation experiments we were able to demonstrate the protease activity of the p 20 protein of HCV.