Gene expression is associated with virulence in murine macrophages infected with Leptospira spp.

Leptospira genus contains species that affect human health with varying degrees of pathogenicity. In this context, we aimed to evaluate the differences in the modulation of host gene expression by strains of Leptospira varying in virulence. Our data showed a high number of differentially expressed transcripts in murine macrophages following 6h of infection. Leptospira infection modulated a set of genes independently of their degree of virulence. However, pathway analysis indicated that Apoptosis, ATM Signaling, and Cell Cycle: G2/M DNA Damage Checkpoint Regulation were exclusively regulated following infection with the virulent strain. Taken together, results demonstrated that species and virulence play a role during host response to Leptospira spp in murine macrophages, which could contribute to understanding the pathogenesis of leptospirosis.

Characterization of the microtranscriptome of macrophages infected with virulent, attenuated and saprophyte strains of Leptospira spp.

Leptospirosis is a bacterial zoonosis, caused by Leptospira spp., that leads to significant morbidity and mortality worldwide. Despite considerable advances, much is yet to be discovered about disease pathogenicity. The influence of epigenetic mechanisms, particularly RNA-mediated post-transcriptional regulation of host immune response has been described following a variety of bacterial infections. The current study examined the microtranscriptome of macrophages J774A.1 following an 8h infection with virulent, attenuated and saprophyte strains of Leptospira. Microarray analysis revealed that 29 miRNAs were misregulated following leptospiral infection compared to control macrophages in a strain and virulence-specific manner. Pathway analysis for targets of these differentially expressed miRNAs suggests that several processes involved in immune response could be regulated by miRNAs. Our data provides the first evidence that host miRNAs are regulated by Leptospira infection in macrophages. A number of the identified miRNA targets participate in key immune response processes. We suggest that post-transcriptional regulation by miRNAs may play a role in host response to infection in leptospirosis.

A new direct transfer protocol for cryopreserved IVF embryos.

The global demand for in vitro-produced (IVP) embryos of determined sex has greatly increased over the last decade. Efficient protocols for the direct transfer of IVP embryos are lacking. This study aimed to compare the pregnancy rates for fresh, vitrified, or frozen/directly transferred IVP dairy cow embryos. Oocytes (n = 3171) recovered by ovum pickup (n = 112) from Girolando (Holstein-Gir) females (n = 36) were selected and submitted to IVM for 24 hours at 38.5 °C with 5% CO2 in air with saturated humidity. In vitro fertilization was performed with the thawed, sexed semen from 5 Holstein bulls. After IVF, presumptive zygotes were denuded and cultured for 7 days under the same IVM and IVF conditions of temperature and humidity, except with 5% CO2 and 5% O2. Grade I blastocysts were randomly assigned for either the transferred fresh, vitrified/thawing, or frozen/directly embryo transfer into previously synchronized recipient females. Conception rates were analyzed by binomial logistic regression, and a probability level of P < 0.05 was considered significant. The conception rates were 51.35 ± 1.87% (133/259) for the fresh embryos, 35.89 ± 3.87% (84/234) for the vitrified embryos, and 40.19 ± 4.65% (125/311) for the frozen directly transferred embryos. These data demonstrate that IVP embryos with sexed semen could be directly transferred into recipient cows with similar conception rates to vitrified embryos. The comparison found that the use of frozen embryos in direct transfer provides easier logistics and a more practical approach for the transfer of IVP embryos on dairy farms.

Changes in tri-methylation profile of lysines 4 and 27 of histone H3 in bovine blastocysts after cryopreservation.

Pregnancy rates from cryopreserved embryos remain lower than non-cryopreserved counterparts, even though these embryos appear morphologically normal. How epigenetic events, such as histone modifications, are affected by cryopreservation of embryos remains unknown. The current study evaluated the effect of conventional freezing/thawing of in vitro produced bovine blastocyst embryos on histone modifications, H3K4me3 and H3K27me3. At day 7 of in vitro culture, blastocyst stage embryos were either frozen by conventional freezing method (-0.5 °C/min in 1.5 M ethylene glycol; F/T group) or remained in culture for an additional 18 h (Ctrl). Frozen embryos were stored in liquid N2 for 14 days, thawed and placed in culture for 36 h for recovery. Control and re-expanded frozen-thawed blastocysts from both groups were fixed in 4% paraformaldehyde and stored in PBS +0.1% triton-X at 4 °C. Immunofluorescence, utilizing antibodies against H3K4me3 and H3K27me3, was conducted and staining intensity was analyzed as percentage of total DNA. Day 7 blastocyst development rate was 35.55% (352/990) with blastocyst recovery at 54.23% (77/142) 36 h post-thawing. Total cell numbers per blastocyst were not different amongst groups (117.8 ± 12.49 and 116.1 ± 14.69, F/T and Ctrl groups respectively). Global staining for the active mark, H3K4me3, was lower in F/T blastocysts compared to Ctrl (17.24 ± 2.80% vs. 34.95 ± 3.77%; P < 0.01). However, staining for the inhibitory mark, H3K27me3, was nearly 2-fold higher in F/T blastocysts (40.41 ± 3.83% vs. 21.29 ± 3.92%; P < 0.01). These results suggest that bovine blastocysts, subjected to conventional freezing methods, have altered histone modifications that may play a role in poor pregnancy rates.

Chromatin modifying agents in the in vitro production of bovine embryos.

The low efficiency observed in cloning by nuclear transfer is related to an aberrant gene expression following errors in epigenetic reprogramming. Recent studies have focused on further understanding of the modifications that take place in the chromatin of embryos during the preimplantation period, through the use of chromatin modifying agents. The goal of these studies is to identify the factors involved in nuclear reprogramming and to adjust in vitro manipulations in order to better mimic in vivo conditions. Therefore, proper knowledge of epigenetic reprogramming is necessary to prevent possible epigenetic errors and to improve efficiency and the use of in vitro fertilization and cloning technologies in cattle and other species.