Zona-penetration in vitro test for evaluating boar sperm fertility

We investigated the capacity of porcine sperm-zona binding and penetration by using bioassay to differentiate between spermatozoa from fertile and subfertile boars. Semen was collected from Large White boars grouped into categories of fertile and subfertile (n=5 per each group) according to the results of artificial insemination. Boars in both groups showed similarly hyperactivated sperm motility at insemination (44.72 and 43.03% respectively) regardless of the lower percentage of progressive motility observed in the ejaculates of subfertile boars. At in vitro insemination, a high proportion of the sperm population (43.76%) in the subfertile boars was without acrosomes, while in the fertile boars this proportion was only 24.35%. The sperm penetration rate of fertile boars reached 66.03% while that of subfertile boars was only 25.08%. In conclusion, the results of our study showed that the penetration rate by boar spermatozoa of the zona pellucida can be used to predict fertility and/or as an in vitro standard for describing porcine semen characteristics.     

mtDNA diversity in rhesus monkeys reveals overestimates of divergence time and paraphyly with neighboring species

Reconstructions of the human-African great ape phylogeny by using mitochondrial DNA (mtDNA) have been subject to considerable debate. One confounding factor may be the lack of data on intraspecific variation. To test this hypothesis, we examined the effect of intraspecific mtDNA diversity on the phylogenetic reconstruction of another Plio- Pleistocene radiation of higher primates, the fascicularis group of macaque (Macaca) monkey species. Fifteen endonucleases were used to identify 10 haplotypes of 40-47 restriction sites in M. mulatta, which were compared with similar data for the other members of this species group. Interpopulational, intraspecific mtDNA diversity was large (0.5%- 4.5%), and estimates of divergence time and branching order incorporating this variation were substantially different from those based on single representatives of each species. We conclude that intraspecific mtDNA diversity is substantial in at least some primate species. Consequently, without prior information on the extent of genetic diversity within a particular species, intraspecific variation must be assessed and accounted for when reconstructing primate phylogenies. Further, we question the reliability of hominoid mtDNA phylogenies, based as they are on one or a few representatives of each species, in an already depauperate superfamily of primates.      

Biochemical and immunochemical characterization of boar sperm flagellar protein with role in hyperactivation/capacitation process

Investigations on specific and functionally active sperm antigens could bring about the elucidation of the mechanisms of gamete interaction and help the search for new approaches in prognosis and regulation of fertility. Previously, we reported that the monoclonal antibody (Mab) 3G4 against capacitated boar spermatozoa was capable of inhibiting boar sperm-porcine zona pellucida binding due to its inhibitory effect on sperm hyperactivation and capacitation. The cell and tissue specificity of Mab 3G4 was demonstrated in indirect immunofluorescence (IIF) and ELISA experiments against spermatozoa from different vertebrate species, as well as against extracts of boar reproductive and somatic organs. In the present IIF experiments, it was shown that Mab 3G4 recognized an antigen determinant on the flagellar midpiece region of ejaculated and capacitated boar spermatozoa. It was speculated that the Mab 3G4-corresponding antigen participates in pyruvate/lactate metabolism because of its specific localization in the sperm structure, which is responsible for producing forward motility and its involvement in processes that require the metabolism of pyruvate and lactate. As a possible approach toward investigating the participation of Ag 3G4 in pyruvate/lactate metabolism, Mab 3G4's effect on lactate dehydrogenase (LDH) was examined. Using an electrophoretic approach we provided evidence that Mab 3G4 stimulates LDH activity in the Triton X-100 and NP40 protein fractions of capacitated boar spermatozoa. In addition, we found that LDH isoenzymes stimulated by Mab 3G4 are of gametic C type. In Western blot, under nonreducing conditions, Mab 3G4 identified a single protein band with a molecular weight of 140 kDa. The biochemical and immunochemical experiments provided evidence supporting the involvement of 3G4 antigen in the sperm pyruvate/lactate metabolism.     

NMR solution structure determination of RNAs

During the past several years, there have been significant advances in NMR solution structure determination of macromolecules. The ability to easily measure residual dipolar couplings, to directly detect NHellipsisN hydrogen bonding interactions and to study much larger macromolecules by the application of heteronuclear experiments that select narrow lines in 2D and 3D spectra of isotopically labeled molecules promises to dramatically improve solution structure determination of nucleic acids.     

Complex evaluation of human monocyte‐derived dendritic cells for cancer immunotherapy

Dendritic cell (DC) immunotherapy is capable of generating tumour‐specific immune responses. Different maturation strategies were previously tested to obtain DC capable of anti‐cancer responses in vitro, usually with limited clinical benefit. Mutual comparison of currently used maturation strategies and subsequent complex evaluation of DC functions and their stimulatory capacity on T cells was performed in this study to optimize the DC vaccination strategy for further clinical application. DC were generated from monocytes using granulocyte–macrophage colony‐stimulating factor (GM‐CSF) and interleukin (IL)‐4, pulsed with whole tumour cell lysate and then matured with one of five selected maturation strategies or cultured without additional maturation stimulus. DC were characterized with regard to their surface marker expression, cytokine profiles, migratory capacity, allogeneic and autologous T cell stimulatory capacity as well as their specific cytotoxicity against tumour antigens. We were able to demonstrate extensive variability among different maturation strategies currently used in DC immunotherapeutic protocols that may at least partially explain limited clinical benefit of some clinical trials with such DC. We identified DC matured with interferon‐γ and lipopolysaccharide as the most attractive candidate for future clinical trials in cancer immunotherapy.     

Ultrastructural study of cat zona pellucida during oocyte maturation and fertilization

The mammalian zona pellucida (ZP) is an extracellular glycoprotein structure formed around growing oocytes, ovulated eggs and preimplantation embryos. The specific functions of ZP are highly determined by its morphological structure. Studies on cat oocytes during maturation and after fertilization were undertaken, using routine transmission (TEM) and scanning electron microscopy (SEM). Two basic ZP layers – outer with rough spongy appearance and inner with smaller fenestrations and smooth fibrous network – were visible. Deposits, secreted by oviductal cells formed new layer, the so called oviductal ZP. After fertilization outer ZP showed rougher meshed network due to fusion between filaments as a consequence from sperm penetration while the inner was smoother with melted appearance. The presented data on the SEM and TEM characteristics of cat oocytes, together with our previous studies on carbohydrate distribution suggest that during oocyte maturation and fertilization ZP undergoes structural and functional rearrangements related to sperm binding and penetration.     

Novel,potent, selective,and metabolically stable stearoyl-CoA desaturase (SCD) inhibitors

We identified a series of structurally novel SCD (Δ9 desaturase) inhibitors via high-throughput screening and follow-up SAR studies. Modification of the central bicyclic scaffold has proven key to our potency optimization effort. The most potent analog (8g) had IC₅₀ value of 50 pM in a HEPG2 SCD assay and has been shown to be metabolically stable and selective against Δ5 and Δ6 desaturases.