First sex modification case in equine cloning.

Somatic cell nuclear transfer (SCNT) is an asexual reproductive technique where cloned offspring contain the same genetic material as the original donor. Although this technique preserves the sex of the original animal, the birth of sex-reversed offspring has been reported in some species. Here, we report for the first time the birth of a female foal generated by SCNT of a male nuclear donor. After a single SCNT procedure, 16 blastocysts were obtained and transferred to eight recipient mares, resulting in the birth of two clones: one male and one female. Both animals had identical genetic profiles, as observed in the analysis of 15-horse microsatellite marker panel, which confirmed they are indeed clones of the same animal. Cytogenetic analysis and fluorescent in situ hybridization using X and Y specific probes revealed a 63,X chromosome set in the female offspring, suggesting a spontaneous Y chromosome loss. The identity of the lost chromosome in the female was further confirmed through PCR by observing the presence of X-linked markers and absence of Y-linked markers. Moreover, cytogenetic and molecular profiles were analyzed in blood and skin samples to detect a possible mosaicism in the female, but results showed identical chromosomal constitutions. Although the cause of the spontaneous chromosome loss remains unknown, the possibility of equine sex reversal by SCNT holds great potential for the preservation of endangered species, development of novel breeding techniques, and sportive purposes.

Generation of myostatin edited horse embryos using CRISPR/Cas9 technology and somatic cell nuclear transfer.

The application of new technologies for gene editing in horses may allow the generation of improved sportive individuals. Here, we aimed to knock out the myostatin gene (MSTN), a negative regulator of muscle mass development, using CRISPR/Cas9 and to generate edited embryos for the first time in horses. We nucleofected horse fetal fibroblasts with 1, 2 or 5 µg of 2 different gRNA/Cas9 plasmids targeting the first exon of MSTN. We observed that increasing plasmid concentrations improved mutation efficiency. The average efficiency was 63.6% for gRNA1 (14/22 edited clonal cell lines) and 96.2% for gRNA2 (25/26 edited clonal cell lines). Three clonal cell lines were chosen for embryo generation by somatic cell nuclear transfer: one with a monoallelic edition, one with biallelic heterozygous editions and one with a biallelic homozygous edition, which rendered edited blastocysts in each case. Both MSTN editions and off-targets were analyzed in the embryos. In conclusion, CRISPR/Cas9 proved an efficient method to edit the horse genome in a dose dependent manner with high specificity. Adapting this technology sport advantageous alleles could be generated, and a precision breeding program could be developed.

MicroRNA characterization in equine induced pluripotent stem cells.

Cell reprogramming has been well described in mouse and human cells. The expression of specific microRNAs has demonstrated to be essential for pluripotent maintenance and cell differentiation, but not much information is available in domestic species. We aim to generate horse iPSCs, characterize them and evaluate the expression of different microRNAs (miR-302a,b,c,d, miR-205, miR-145, miR-9, miR-96, miR-125b and miR-296). Two equine iPSC lines (L2 and L3) were characterized after the reprogramming of equine fibroblasts with the four human Yamanaka's factors (OCT-4/SOX-2/c-MYC/KLF4). The pluripotency of both lines was assessed by phosphatase alkaline activity, expression of OCT-4, NANOG and REX1 by RT-PCR, and by immunofluorescence of OCT-4, SOX-2 and c-MYC. In vitro differentiation to embryo bodies (EBs) showed the capacity of the iPSCs to differentiate into ectodermal, endodermal and mesodermal phenotypes. MicroRNA analyses resulted in higher expression of the miR-302 family, miR-9 and miR-96 in L2 and L3 vs. fibroblasts (p<0.05), as previously shown in human pluripotent cells. Moreover, downregulation of miR-145 and miR-205 was observed. After differentiation to EBs, higher expression of miR-96 was observed in the EBs respect to the iPSCs, and also the expression of miR-205 was induced but only in the EB-L2. In addition, in silico alignments of the equine microRNAs with mRNA targets suggested the ability of miR-302 family to regulate cell cycle and epithelial mesenchymal transition genes, miR-9 and miR-96 to regulate neural determinant genes and miR-145 to regulate pluripotent genes, similarly as in humans. In conclusion, we could obtain equine iPSCs, characterize them and determine for the first time the expression level of microRNAs in equine pluripotent cells.

In Vitro and In Vivo Development of Horse Cloned Embryos Generated with iPSCs, Mesenchymal Stromal Cells and Fetal or Adult Fibroblasts as Nuclear Donors.

The demand for equine cloning as a tool to preserve high genetic value is growing worldwide; however, nuclear transfer efficiency is still very low. To address this issue, we first evaluated the effects of time from cell fusion to activation (<1h, n = 1261; 1-2h, n = 1773; 2-3h, n = 1647) on in vitro and in vivo development of equine embryos generated by cloning. Then, we evaluated the effects of using different nuclear donor cell types in two successive experiments: I) induced pluripotent stem cells (iPSCs) vs. adult fibroblasts (AF) fused to ooplasts injected with the pluripotency-inducing genes OCT4, SOX2, MYC and KLF4, vs. AF alone as controls; II) umbilical cord-derived mesenchymal stromal cells (UC-MSCs) vs. fetal fibroblasts derived from an unborn cloned foetus (FF) vs. AF from the original individual. In the first experiment, both blastocyst production and pregnancy rates were higher in the 2-3h group (11.5% and 9.5%, respectively), respect to <1h (5.2% and 2%, respectively) and 1-2h (5.6% and 4.7%, respectively) groups (P<0.05). However, percentages of born foals/pregnancies were similar when intervals of 2-3h (35.2%) or 1-2h (35.7%) were used. In contrast to AF, the iPSCs did not generate any blastocyst-stage embryos. Moreover, injection of oocytes with the pluripotency-inducing genes did not improve blastocyst production nor pregnancy rates respect to AF controls. Finally, higher blastocyst production was obtained using UC-MSC (15.6%) than using FF (8.9%) or AF (9.3%), (P<0.05). Despite pregnancy rates were similar for these 3 groups (17.6%, 18.2% and 22%, respectively), viable foals (two) were obtained only by using FF. In summary, optimum blastocyst production rates can be obtained using a 2-3h interval between cell fusion and activation as well as using UC-MSCs as nuclear donors. Moreover, FF line can improve the efficiency of an inefficient AF line. Overall, 24 healthy foals were obtained from a total of 29 born foals.

Improvement of bovine semen quality by removal of membrane-damaged sperm cells with DNA aptamers and magnetic nanoparticles.

In cattle, cryopreservation of semen and sex-sorting kill up to 50% of spermatozoa and decrease the success of assisted insemination (AI). Therefore, significant efforts are being carried out to improve the quality of semen prior to AI. In this work we used the Cell-SELEX technique to select single strand DNA aptamers able to recognize with high affinity and specificity damaged sperm cells generated by heat-treatment. We first isolated aptamers with a conserved two motifs of 6 nucleotides of length that bind to the membrane of heat-treated spermatozoa. Then, we used synthetic biotin-labeled aptamers containing the conserved motif to recognize membrane-damaged cells and separate them from viable cells by the use of avidin-coated superparamagnetic iron oxide nanoparticles (SPION). This procedure improved the quality of semen by significantly increasing the percentage of healthy sperm cells without affecting the rate of blastocyst cleavage. This technique was successfully applied to both unsorted and sex-sorted sperm suspension.

Oocyte genome cloning used in biparental bovine embryo reconstruction.

Oocyte genome cloning is a method by which haploid maternal embryos are obtained in such a way that parthenogenetic haploid blastomeres from these embryos can be considered as a clone of the original gamete. Our objective was to generate oocyte genome replicates and use them to reconstruct biparental embryos by fusion with haploid male hemizygotes. Furthermore, we generated biparental homogeneous transgene-expressing embryos using parthenogenetic haploid blastomeres that expressed a transgene (EGFP). In the first experiment, parthenogenetic haploid embryos were generated by incubation of oocytes in ionomycin and 6-dimethylaminopurine (DMAP) with a 3 h interval to permit their second polar body extrusion. The cleavage rate was 87.3%. To generate transgene-expressing blastomeres, activated oocytes were injected with pCX-EGFP-liposome complexes 3 h post ionomycin exposure, resulting in a cleavage rate of 84.4%. In the second experiment, haploid parthenogenetic blastomeres that were positive or negative for EGFP expression were used to reconstruct biparental embryos. Cleavage and blastocyst rates for the reconstructed embryos were 78.4% and 61.1% and 10.8% and 8.4%, using EGFP-positive or -negative blastomeres, respectively (P < 0.05). All of the reconstructed embryos showed EGFP expression, with 96.6% of them showing homogenic expression. Oct-4 expression in the reconstructed blastocysts displayed a similar pattern as IVF-blastocyst controls. In conclusion, our results proved that it is possible to use oocyte genome replicates to reconstruct biparental bovine embryos and that this technique is efficient to generate homogeneous transgene-expressing embryos.

Sperm genome cloning used in biparental bovine embryo reconstruction.

The generation of androgenetic haploid embryos enables several haploid blastomeres to be obtained as identical copies of a single spermatozoon genome. In the present study, we compared the developmental ability of bovine androgenetic haploid embryos constructed by different methods, namely IVF and intracytoplasmic sperm injection (ICSI) before and after oocyte enucleation. Once obtained, the blastomeres of these androgenetic haploid embryos were used as male genome donors to reconstruct biparental embryos by fusion with matured oocytes. To verify the cytoplasmic contribution of androgenetic haploid blastomeres, we used spermatozoa incubated previously with exogenous DNA that coded for a green fluorescent protein gene (pCX-EGFP) and the enhanced green fluorescent protein (EGFP)-positive androgenetic haploid blastomeres generated were fused with mature oocytes. Of the reconstructed embryos reaching the cleavage and blastocyst stages, 85.1% and 9.0%, respectively, expressed EGFP (P>0.05). EGFP expression was observed in 100% of reconstructed embryos, with 91.2% exhibiting homogenic expression. To confirm sperm genome incorporation, androgenetic haploid blastomeres generated by ICSI prior to enucleation and using Y chromosome sexed spermatozoa were used for biparental embryo reconstruction. Incorporation of the Y chromosome was confirmed by polymerase chain reaction and fluorescence in situ hybridisation analysis. In conclusion, the results of the present study prove that it is possible to use sperm genome replicates to reconstruct biparental bovine embryos and that it is a highly efficient technique to generate homogeneous transgene-expressing embryos.

Suppression of COX-2, IL-1ß and TNF-α expression and leukocyte infiltration in inflamed skin by bioactive compounds from Rosmarinus officinalis L.

In the present study, we evaluated the effects of extracts and purified compounds from fresh leaves of Rosmarinus officinalis L. Pretreatment with the major anti-inflammatory compounds, carnosic acid (CA) and carnosol (CS), inhibited phorbol 12-myristate 13-acetate (PMA)-induced ear inflammation in mice with an EC(50) of 10.20 µg/cm(2) and 10.70 µg/cm(2), respectively. To further understand the anti-inflammatory mechanism of these compounds, we analyzed the in vivo expression of several inflammation-associated genes in mouse skin by reverse transcriptase-polymerase chain reaction (RT-PCR). Our data showed that CA and CS reduced the expression of IL-1ß and TNF-α but had less effect on fibronectin and ICAM-1 expression. Interestingly, both compounds selectively inhibited COX-2 but not COX-1. Histopathological analysis of hematoxylin and eosin (H&E)-stained tissue revealed a marked reduction in leukocyte infiltration and epidermal ulceration of PMA-treated ears when ears were pretreated with ethanolic extracts or pure CA. In vitro, we showed that ethanolic extract, carnosic acid and carnosol significantly inhibited the overproduction of nitric oxide (NO) in a dose-dependent manner in the RAW 264.7 murine macrophage cell line. For the first time in vivo, we showed that CA and CS differentially regulate the expression of inflammation-associated genes, thus demonstrating the pharmacological basis for the anti-inflammatory properties reported for CA and CS.