Optimising Electroporation Condition for CRISPR/Cas-Mediated Knockout in Zona-Intact Buffalo Zygotes.

Somatic cell nuclear transfer or cytoplasm microinjection has widely been used to produce genome-edited farm animals; however, these methods have several drawbacks which reduce their efficiency. In the present study, we describe an easy adaptable approach for the introduction of mutations using CRISPR-Cas9 electroporation of zygote (CRISPR-EP) in buffalo. The goal of the study was to determine the optimal conditions for an experimental method in which the CRISPR/Cas9 system is introduced into in vitro-produced buffalo zygotes by electroporation. Electroporation was performed using different combinations of voltage, pulse and time, and we observed that the electroporation in buffalo zygote at 20 V/mm, 5 pulses, 3 msec at 10 h post insemination (hpi) resulted in increased membrane permeability and higher knockout efficiency without altering embryonic developmental potential. Using the above parameters, we targeted buffalo POU5F1 gene as a proof of concept and found no variations in embryonic developmental competence at cleavage or blastocyst formation rate between control, POU5F1-KO, and electroporated control (EC) embryos. To elucidate the effect of POU5F1-KO on other pluripotent genes, we determined the relative expression of SOX2, NANOG, and GATA2 in the control (POU5F1 intact) and POU5F1-KO-confirmed blastocyst. POU5F1-KO significantly (p ≤ 0.05) altered the expression of SOX2, NANOG, and GATA2 in blastocyst stage embryos. In conclusion, we standardized an easy and straightforward protocol CRISPR-EP method that could be served as a useful method for studying the functional genomics of buffalo embryos.

Low-density lipoproteins protect sperm during cryopreservation in buffalo: Unraveling mechanism of action.

This study was carried out to reveal factors and the mechanism of action by which low-density lipoproteins (LDLs) protect sperm better than egg yolk (EY) during cryopreservation. We extracted LDL from EY and compared the amount of calcium, progesterone, and antioxidants in EY and LDL. We found a very high concentration of progesterone (1423.95 vs. 10.46 ng/ml) and calcium (29.19 vs. 0.47 mM) in EY as compared with LDL. Antioxidant assays like DPPH (2,2-diphenyl-1-picrylhydrazyl) and the ferric reducing antioxidants power assay revealed that the LDL extender had almost double ability to lose hydrogen than the EY extender. For sperm cryopreservation, 20 ejaculates from four Murrah buffalo bulls were collected. Each ejaculate was divided into four aliquots and extended in 10%, 12%, and 14% LDL (w/v) and EY-based extenders, followed by cryopreservation. The LDL-based extender prevented excessive cholesterol efflux, and its high content of antioxidants minimized reactive oxygen species generated during cryopreservation, resulting in a functional CatSper channel. The EY-based extender promoted excess cholesterol efflux due to the presence of high-density lipoprotein, resulting in a compromised CatSper channel. High intracellular calcium in a cryopreserved sperm in the EY group as compared with the LDL group indicates that progesterone present in EY activates the CatSper channel, resulting in a heavy calcium influx into the sperm. The greater tyrosine phosphorylation and increased number of F-pattern in the sperm cryopreserved in the EY extender indicate that high intracellular calcium triggers more capacitation-like changes in the sperm cryopreserved in EY than LDL extender. In conclusion, we demonstrated the new facts and understandings about LDL and EY for semen cryopreservation.

The domesticated buffalo - An emerging model for experimental and therapeutic use of extraembryonic tissues.

Large animals play important roles as model animals for biomedical sciences and translational research. The water buffalo (Bubalus bubalis) is an economically important, multipurpose livestock species. Important assisted reproduction techniques, such as in vitro fertilization, cryo-conservation of sperm and embryos, embryo transfer, somatic cell nuclear transfer, genetic engineering, and genome editing have been successfully applied to buffaloes. Recently, detailed whole genome data and transcriptome maps have been generated. In addition, rapid progress has been made in stem cell biology of the buffalo. Apart from embryonic stem cells, bubaline extra-embryonic stem cells have gained particular interest. The multipotency of non-embryonic stem cells has been revealed, and their utility in basic and applied research is currently investigated. In particular, success achieved in bubaline extra-embryonic stem cells may have important roles in experimental biology and therapeutic regenerative medicine. Progress in other farm animals in assisted reproduction techniques, stem cell biology and genetic engineering, which could be of importance for buffalo, will also be briefly summarized.

Piezo1/2 mediate mechanotransduction essential for bone formation through concerted activation of NFAT-YAP1-ß-catenin.

Mechanical forces are fundamental regulators of cell behaviors. However, molecular regulation of mechanotransduction remain poorly understood. Here, we identified the mechanosensitive channels Piezo1 and Piezo2 as key force sensors required for bone development and osteoblast differentiation. Loss of Piezo1, or more severely Piezo1/2, in mesenchymal or osteoblast progenitor cells, led to multiple spontaneous bone fractures in newborn mice due to inhibition of osteoblast differentiation and increased bone resorption. In addition, loss of Piezo1/2 rendered resistant to further bone loss caused by unloading in both bone development and homeostasis. Mechanistically, Piezo1/2 relayed fluid shear stress and extracellular matrix stiffness signals to activate Ca2+ influx to stimulate Calcineurin, which promotes concerted activation of NFATc1, YAP1 and ß-catenin transcription factors by inducing their dephosphorylation as well as NFAT/YAP1/ß-catenin complex formation. Yap1 and ß-catenin activities were reduced in the Piezo1 and Piezo1/2 mutant bones and such defects were partially rescued by enhanced ß-catenin activities.

Bone morphogenetic protein-12 induces tenogenic differentiation of mesenchymal stem cells derived from equine amniotic fluid.

Tendon injuries are common in race horses, and mesenchymal stem cells (MSCs) isolated from adult and foetal tissue have been used for tendon regeneration. In the present study, we evaluated equine amniotic fluid (AF) as a source of MSCs and standardised methodology and markers for their in vitro tenogenic differentiation. Plastic-adherent colonies were isolated from 12 of 20 AF samples by day 6 after seeding and 70-80% cell confluency was reached by day 17. These cells expressed mesenchymal surface markers [cluster of differentiation (CD)73, CD90 and CD105] by reverse transcription (RT)-polymerase chain reaction (PCR) and immunocytochemistry, but did not express haematopoietic markers (CD34, CD45 and CD14). In flow cytometry, the expression of CD29, CD44, CD73 and CD90 was observed in 68.83 ± 1.27, 93.66 ± 1.80, 96.96 ± 0.44 and 93.7 ± 1.89% of AF-MSCs, respectively. Osteogenic, chondrogenic and adipogenic differentiation of MSCs was confirmed by von Kossa and Alizarin red S, Alcian blue and oil red O staining, respectively. Upon supplementation of MSC growth media with 50 ng/ml bone morphogenetic protein (BMP)-12, AF-MSCs differentiated to tenocytes within 14 days. The differentiated cells were more slender, elongated and spindle shaped with thinner and longer cytoplasmic processes and showed expression of tenomodulin and decorin by RT-PCR and immunocytochemistry. In flow cytometry, 96.7 ± 1.90 and 80.9 ± 6.4% of differentiated cells expressed tenomodulin and decorin in comparison to 1.6 and 3.1% in undifferentiated control cells, respectively. Our results suggest that AF is an easily accessible and effective source of MSCs. On BMP-12 supplementation, AF-MSCs can be differentiated to tenocytes, which could be exploited for regeneration of ruptured or damaged tendon in race horses.

Bovine ICM derived cells express the Oct4 ortholog.

The goal of this study was to define conditions for the successful isolation of embryonic stem cells from bovine blastocysts. Expression of the Pit-Oct-Unc (POU) transcription factor Oct4 was employed to monitor the pluripotent status of cultured cells. No expression of the previously identified bovine Oct4 pseudogene was found, and transcription of the Oct4 ortholog correlated with the proliferative potential of bovine ICM derived cells. Two methods to isolate pluripotent inner cell mass were compared; 90% of trypsin isolated ICMs formed growing cultures, whereas only 12%-23% of the ICMs isolated by immunosurgery attached and grew. Colony formation from complete blastocysts was 55%. The bovine ICM derived cells could be grown for 4-7 passages. However, Oct4 transcripts were only present in the primary cultures, indicating that the initial culture period of bovine ICM derived cells is critical and needs to be optimized to yield true ES cells. In contrast to bovine ICMs, murine ICMs yielded rapidly growing cells, which proliferated for more than 60 passages.