Discovery of ciliary G protein-coupled receptors regulating pancreatic islet insulin and glucagon secretion.

Multiple G protein-coupled receptors (GPCRs) are expressed in pancreatic islet cells, but the majority have unknown functions. We observed specific GPCRs localized to primary cilia, a prominent signaling organelle, in pancreatic α and ß cells. Loss of cilia disrupts ß-cell endocrine function, but the molecular drivers are unknown. Using functional expression, we identified multiple GPCRs localized to cilia in mouse and human islet α and ß cells, including FFAR4, PTGER4, ADRB2, KISS1R, and P2RY14. Free fatty acid receptor 4 (FFAR4) and prostaglandin E receptor 4 (PTGER4) agonists stimulate ciliary cAMP signaling and promote glucagon and insulin secretion by α- and ß-cell lines and by mouse and human islets. Transport of GPCRs to primary cilia requires TULP3, whose knockdown in primary human and mouse islets relocalized ciliary FFAR4 and PTGER4 and impaired regulated glucagon or insulin secretion, without affecting ciliary structure. Our findings provide index evidence that regulated hormone secretion by islet α and ß cells is controlled by ciliary GPCRs providing new targets for diabetes.

SIX2 and SIX3 coordinately regulate functional maturity and fate of human pancreatic ß cells.

The physiological functions of many vital tissues and organs continue to mature after birth, but the genetic mechanisms governing this postnatal maturation remain an unsolved mystery. Human pancreatic ß cells produce and secrete insulin in response to physiological cues like glucose, and these hallmark functions improve in the years after birth. This coincides with expression of the transcription factors SIX2 and SIX3, whose functions in native human ß cells remain unknown. Here, we show that shRNA-mediated SIX2 or SIX3 suppression in human pancreatic adult islets impairs insulin secretion. However, transcriptome studies revealed that SIX2 and SIX3 regulate distinct targets. Loss of SIX2 markedly impaired expression of genes governing ß-cell insulin processing and output, glucose sensing, and electrophysiology, while SIX3 loss led to inappropriate expression of genes normally expressed in fetal ß cells, adult α cells, and other non-ß cells. Chromatin accessibility studies identified genes directly regulated by SIX2. Moreover, ß cells from diabetic humans with impaired insulin secretion also had reduced SIX2 transcript levels. Revealing how SIX2 and SIX3 govern functional maturation and maintain developmental fate in native human ß cells should advance ß-cell replacement and other therapeutic strategies for diabetes.

Molecular and genetic regulation of pig pancreatic islet cell development.

Reliance on rodents for understanding pancreatic genetics, development and islet function could limit progress in developing interventions for human diseases such as diabetes mellitus. Similarities of pancreas morphology and function suggest that porcine and human pancreas developmental biology may have useful homologies. However, little is known about pig pancreas development. To fill this knowledge gap, we investigated fetal and neonatal pig pancreas at multiple, crucial developmental stages using modern experimental approaches. Purification of islet ß-, α- and δ-cells followed by transcriptome analysis (RNA-seq) and immunohistology identified cell- and stage-specific regulation, and revealed that pig and human islet cells share characteristic features that are not observed in mice. Morphometric analysis also revealed endocrine cell allocation and architectural similarities between pig and human islets. Our analysis unveiled scores of signaling pathways linked to native islet ß-cell functional maturation, including evidence of fetal α-cell GLP-1 production and signaling to ß-cells. Thus, the findings and resources detailed here show how pig pancreatic islet studies complement other systems for understanding the developmental programs that generate functional islet cells, and that are relevant to human pancreatic diseases.

Baculovirus Transduction in Mammalian Cells Is Affected by the Production of Type I and III Interferons, Which Is Mediated Mainly by the cGAS-STING Pathway.

The baculovirus Autographa californica multiple nucleopolyhedrovirus is an insect virus with a circular double-stranded DNA genome, which, among other multiple biotechnological applications, is used as an expression vector for gene delivery in mammalian cells. Nevertheless, the nonspecific immune response triggered by viral vectors often suppresses transgene expression. To understand the mechanisms involved in that response, in the present study, we studied the cyclic GMP-AMP synthase-stimulator of interferon genes (cGAS-STING) pathway by using two approaches: the genetic edition through CRISPR/Cas9 technology of genes encoding STING or cGAS in NIH/3T3 murine fibroblasts and the infection of HEK293 and HEK293 T human epithelial cells, deficient in cGAS and in cGAS and STING expression, respectively. Overall, our results suggest the existence of two different pathways involved in the establishment of the antiviral response, both dependent on STING expression. Particularly, the cGAS-STING pathway resulted in the more relevant production of beta interferon (IFN-ß) and IFN-λ1 in response to baculovirus infection. In human epithelial cells, IFN-λ1 production was also induced in a cGAS-independent and DNA-protein kinase (DNA-PK)-dependent manner. Finally, we demonstrated that these cellular responses toward baculovirus infection affect the efficiency of transduction of baculovirus vectors.IMPORTANCE Baculoviruses are nonpathogenic viruses that infect mammals, which, among other applications, are used as vehicles for gene delivery. Here, we demonstrated that the cytosolic DNA sensor cGAS recognizes baculoviral DNA and that the cGAS-STING axis is primarily responsible for the attenuation of transduction in human and mouse cell lines through type I and type III IFNs. Furthermore, we identified DNA-dependent protein kinase (DNA-PK) as a cGAS-independent and alternative DNA cytosolic sensor that contributes less to the antiviral state in baculovirus infection in human epithelial cells than cGAS. Knowledge of the pathways involved in the response of mammalian cells to baculovirus infection will improve the use of this vector as a tool for gene therapy.

CRISPR-on for activation of endogenous SMARCA4 and TFAP2C expression in bovine embryos.

CRISPR-mediated transcriptional activation, also known as CRISPR-on, has proven efficient for activation of individual or multiple endogenous gene expression in cultured cells from several species. However, the potential of CRISPR-on technology in preimplantation mammalian embryos remains to be explored. Here, we report for the first time the successful modulation of endogenous gene expression in bovine embryos by using the CRISPR-on system. As a proof of principle, we targeted the promoter region of either SMARCA4 or TFAP2C genes, transcription factors implicated in trophoblast lineage commitment during embryo development. We demonstrate that CRISPR-on provides temporal control of endogenous gene expression in bovine embryos, by simple cytoplasmic injection of CRISPR RNA components into one cell embryos. dCas9VP160 activator was efficiently delivered and accurately translated into protein, being detected in the nucleus of all microinjected blastomeres. Our approach resulted in the activation of SMARCA expression shortly after microinjection, with a consequent effect on downstream differentiation promoting factors, such as TFAP2C and CDX2. Although targeting of TFAP2C gene did not result in a significant increase in TFAP2C expression, there was a profound induction in CDX2 expression on day 2 of development. Finally, we demonstrate that CRISPR-on system is suitable for gene expression modulation during the preimplantation period, since no detrimental effect was observed on microinjected embryo development. This study constitutes a first step toward the application of the CRISPR-on system for the study of early embryo cell fate decisions in cattle and other mammalian embryos, as well as to design novel strategies that may lead to an improved trophectoderm development.

Crotamine, a cell-penetrating peptide, is able to translocate parthenogenetic and in vitro fertilized bovine embryos but does not improve exogenous DNA expression.

PURPOSE: Crotamine is capable of penetrating cells and embryos and transfecting cells with exogenous DNA. However, no studies are available regarding its uptake by parthenogenetic (PA) embryos or its use for transfection in in vitro fertilized (IVF) embryos. This study aimed to determine the translocation kinetics of crotamine into PA and IVF bovine embryos and assess its effect over in vitro development of PA embryos. Moreover, crotamine-DNA complexes were used to test the transfection ability of crotamine in bovine IVF zygotes. METHODS: PA and IVF embryos were exposed to labeled crotamine for four interval times. Embryo toxicity was assayed over PA embryos after 24 h of exposure to crotamine. Additionally, IVF embryos were exposed to or injected with a complex formed by crotamine and pCX-EGFP plasmid. RESULTS: Confocal images revealed that crotamine was uptaken by PA and IVF embryos as soon as 1 h after exposure. Crotamine exposure did not affect two to eight cells and blastocyst rates or blastocyst cell number (p > 0.05) of PA embryos. Regarding transfection, exposure or injection into the perivitelline space with crotamine-DNA complex did not result in transgene-expressing embryos. Nevertheless, intracytoplasmic injection of plasmid alone showed higher expression rates than did injection with crotamine-DNA complex at days 4 and 7 (p < 0.05). CONCLUSIONS: Crotamine is able to translocate through zona pellucida (ZP) of PA and IVF embryos within 1 h of exposure without impairing in vitro development. However, the use of crotamine does not improve exogenous DNA expression in cattle embryos, probably due to the tight complexation of DNA with crotamine.

Multiplexed CRISPR gene editing in primary human islet cells with Cas9 ribonucleoprotein.

Successful genome editing in primary human islets could reveal features of the genetic regulatory landscape underlying ß cell function and diabetes risk. Here, we describe a CRISPR-based strategy to interrogate functions of predicted regulatory DNA elements using electroporation of a complex of Cas9 ribonucleoprotein (Cas9 RNP) and guide RNAs into primary human islet cells. We successfully targeted coding regions including the PDX1 exon 1, and non-coding DNA linked to diabetes susceptibility. CRISPR-Cas9 RNP approaches revealed genetic targets of regulation by DNA elements containing candidate diabetes risk SNPs, including an in vivo enhancer of the MPHOSPH9 gene. CRISPR-Cas9 RNP multiplexed targeting of two cis-regulatory elements linked to diabetes risk in PCSK1, which encodes an endoprotease crucial for Insulin processing, also demonstrated efficient simultaneous editing of PCSK1 regulatory elements, resulting in impaired ß cell PCSK1 regulation and Insulin secretion. Multiplex CRISPR-Cas9 RNP provides powerful approaches to investigate and elucidate human islet cell gene regulation in health and diabetes.

RFX6 Maintains Gene Expression and Function of Adult Human Islet α-Cells.

Mutations in the gene encoding the transcription factor regulatory factor X-box binding 6 (RFX6) are associated with human diabetes. Within pancreatic islets, RFX6 expression is most abundant in islet α-cells, and α-cell RFX6 expression is altered in diabetes. However, the roles of RFX6 in regulating gene expression, glucagon output, and other crucial human adult α-cell functions are not yet understood. We developed a method for selective genetic targeting of human α-cells and assessed RFX6-dependent α-cell function. RFX6 suppression with RNA interference led to impaired α-cell exocytosis and dysregulated glucagon secretion in vitro and in vivo. By contrast, these phenotypes were not observed with RFX6 suppression across all islet cells. Transcriptomics in α-cells revealed RFX6-dependent expression of genes governing nutrient sensing, hormone processing, and secretion, with some of these exclusively expressed in human α-cells. Mapping of RFX6 DNA-binding sites in primary human islet cells identified a subset of direct RFX6 target genes. Together, these data unveil RFX6-dependent genetic targets and mechanisms crucial for regulating adult human α-cell function.

Multiplexed CRISPR gene editing in primary human islet cells with Cas9 ribonucleoprotein.

Successful genome editing in primary human islets could reveal features of the genetic regulatory landscape underlying ß cell function and diabetes risk. Here, we describe a CRISPR-based strategy to interrogate functions of predicted regulatory DNA elements using electroporation of a complex of Cas9 ribonucleoprotein (Cas9 RNP) and guide RNAs into primary human islet cells. We successfully targeted coding regions including the PDX1 exon 1, and non-coding DNA linked to diabetes susceptibility. CRISPR/Cas9 RNP approaches revealed genetic targets of regulation by DNA elements containing candidate diabetes risk SNPs, including an in vivo enhancer of the MPHOSPH9 gene. CRISPR/Cas9 RNP multiplexed targeting of two cis-regulatory elements linked to diabetes risk in PCSK1, which encodes an endoprotease crucial for insulin processing, also demonstrated efficient simultaneous editing of PCSK1 regulatory elements, resulting in impaired ß cell PCSK1 regulation and insulin secretion. Multiplex CRISPR/Cas9 RNP provides powerful approaches to investigate and elucidate human islet cell gene regulation in health and diabetes.

HNF1α maintains pancreatic α and ß cell functions in primary human islets.

HNF1A haploinsufficiency underlies the most common form of human monogenic diabetes (HNF1A-maturity onset diabetes of the young [HNF1A-MODY]), and hypomorphic HNF1A variants confer type 2 diabetes risk. But a lack of experimental systems for interrogating mature human islets has limited our understanding of how the transcription factor HNF1α regulates adult islet function. Here, we combined conditional genetic targeting in human islet cells, RNA-Seq, chromatin mapping with cleavage under targets and release using nuclease (CUT&RUN), and transplantation-based assays to determine HNF1α-regulated mechanisms in adult human pancreatic α and ß cells. Short hairpin RNA-mediated (shRNA-mediated) suppression of HNF1A in primary human pseudoislets led to blunted insulin output and dysregulated glucagon secretion after transplantation in mice, recapitulating phenotypes observed in patients with diabetes. These deficits corresponded with altered expression of genes encoding factors critical for hormone secretion, including calcium channel subunits, ATPase transporters, and extracellular matrix constituents. Additionally, HNF1A loss led to upregulation of transcriptional repressors, providing evidence for a mechanism of transcriptional derepression through HNF1α. CUT&RUN mapping of HNF1α DNA binding sites in primary human islets imputed a subset of HNF1α-regulated genes as direct targets. These data elucidate mechanistic links between HNF1A loss and diabetic phenotypes in mature human α and ß cells.

A genome-wide CRISPR screen identifies CALCOCO2 as a regulator of beta cell function influencing type 2 diabetes risk.

Identification of the genes and processes mediating genetic association signals for complex diseases represents a major challenge. As many of the genetic signals for type 2 diabetes (T2D) exert their effects through pancreatic islet-cell dysfunction, we performed a genome-wide pooled CRISPR loss-of-function screen in a human pancreatic beta cell line. We assessed the regulation of insulin content as a disease-relevant readout of beta cell function and identified 580 genes influencing this phenotype. Integration with genetic and genomic data provided experimental support for 20 candidate T2D effector transcripts including the autophagy receptor CALCOCO2. Loss of CALCOCO2 was associated with distorted mitochondria, less proinsulin-containing immature granules and accumulation of autophagosomes upon inhibition of late-stage autophagy. Carriers of T2D-associated variants at the CALCOCO2 locus further displayed altered insulin secretion. Our study highlights how cellular screens can augment existing multi-omic efforts to support mechanistic understanding and provide evidence for causal effects at genome-wide association studies loci.

Novel methods to induce exogenous gene expression in SCNT, parthenogenic and IVF preimplantation bovine embryos.

The import of exogenous DNA (eDNA) from the cytoplasm to the nucleus represents a key intracellular obstacle for efficient gene delivery in mammalian cells. In this study, cumulus cells or oolemma vesicles previously incubated with eDNA, and naked eDNA were injected into the cytoplasm of MII oocytes to evaluate their efficiency for eDNA expressing bovine embryo production. Our study evaluated the potential of short time co-incubation (5 min) of eDNA with; (1) cumulus cells, to be used as donor cells for SCNT and (2) oolemma vesicles (vesicles) to produce parthenogenic transgene expressing embryos. In addition, we included a group consisting of the injection of eDNA alone (plasmid) followed by parthenogenic activation. Two different pCX-EGFP plasmid concentrations (50 and 500 ng/µl) were employed. The results showed that embryos produced by SCNT and by vesicle injection assisted by chemical activation were able to express the eDNA in higher rates than embryos injected with plasmid alone. The lower plasmid concentration allowed the highest development rates in all groups. Using confocal microscopy, we analyzed the interaction of FITC- labeled eDNA with cumulus cells and vesicles as well as oocytes injected with labeled plasmid alone. Our images demonstrated that eDNA interacted with cumulus cells and vesicles, resulting an increase in its expression efficiency. In contrast, oocytes injected with DNA alone did not show signs of transgene accumulation, and their eDNA expression rates were lower. In a further experiment, we evaluated if transgene-expressing embryos could be produced by means of vesicle injection followed by IVF. The lower plasmid concentration (50 ng/µl) injected after IVF, produced the best results. Preliminary FISH analysis indicated detectable integration events in 1/5 of SCNT blastocysts treated. Our studies demonstrate for the first time that short term transgene co-incubation with somatic cells can produce transgene-expressing mammalian SCNT embryos and also that parthenogenic, eDNA- expressing embryos can be obtained by injection of vesicles or eDNA alone. Moreover, eDNA-expressing embryos can be also obtained by cytoplasmic injection vesicles in IVF zygotes, simplifying the traditional IVF pronuclear injection technique.

Bovine parthenogenotes produced by inhibition of first or second polar bodies emission.

Parthenogenetic embryos are an ethically acceptable alternative for the derivation of human embryonic stem cells. In this work, we propose a new strategy to produce bovine parthenogenetic embryos inhibiting the emission of the first polar body during in vitro maturation, and allowing the extrusion of the second polar body during oocyte activation. Cytochalasin B, an inhibitor of actin microfilaments, was employed during in vitro maturation to inhibit first polar body emission or during parthenogenetic activation to block second polar body emission. Only one polar body was inhibited in each strategy in order to keep the diploid chromosome set. In experiment 1, the effect of cytochalasin B on in vitro maturation of bovine oocytes was evaluated. Most oocytes (77%) were arrested at a meiotic stage characterized by the presence of a large internal metaphase plate and absence of polar body. In experiment 2, development of embryos exposed to cytochalasin B during in vitro maturation (CytoB-IVM) or during activation (CytoB-ACT) was compared. Developmental rates did not differ between diploidization strategies, even when three agents were employed to induce activation. Both groups, CytoB-IVM and CytoB-ACT, tended to maintain diploidy. CytoB-IVM parthenogenesis could help to obtain embryos with a higher degree of homology to the oocyte donor.

Pancreatic Pseudoislets: An Organoid Archetype for Metabolism Research.

Pancreatic islets are vital endocrine regulators of systemic metabolism, and recent investigations have increasingly focused on understanding human islet biology. Studies of isolated human islets have advanced understanding of the development, function, and regulation of cells comprising islets, especially pancreatic α- and ß-cells. However, the multicellularity of the intact islet has stymied specific experimental approaches-particularly in genetics and cell signaling interrogation. This barrier has been circumvented by the observation that islet cells can survive dispersion and reaggregate to form "pseudoislets," organoids that retain crucial physiological functions, including regulated insulin and glucagon secretion. Recently, exciting advances in the use of pseudoislets for genetics, genomics, islet cell transplantation, and studies of intraislet signaling and islet cell interactions have been reported by investigators worldwide. Here we review molecular and cellular mechanisms thought to promote islet cell reaggregation, summarize methods that optimize pseudoislet development, and detail recent insights about human islet biology from genetic and transplantation-based pseudoislet experiments. Owing to robust, international programs for procuring primary human pancreata, pseudoislets should serve as both a durable paradigm for primary organoid studies and as an engine of discovery for islet biology, diabetes, and metabolism research.

CRISPR-based genome editing in primary human pancreatic islet cells.

Gene targeting studies in primary human islets could advance our understanding of mechanisms driving diabetes pathogenesis. Here, we demonstrate successful genome editing in primary human islets using clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated protein 9 (Cas9). CRISPR-based targeting efficiently mutated protein-coding exons, resulting in acute loss of islet ß-cell regulators, like the transcription factor PDX1 and the KATP channel subunit KIR6.2, accompanied by impaired ß-cell regulation and function. CRISPR targeting of non-coding DNA harboring type 2 diabetes (T2D) risk variants revealed changes in ABCC8, SIX2 and SIX3 expression, and impaired ß-cell function, thereby linking regulatory elements in these target genes to T2D genetic susceptibility. Advances here establish a paradigm for genetic studies in human islet cells, and reveal regulatory and genetic mechanisms linking non-coding variants to human diabetes risk.

SARS-CoV-2 infects human pancreatic ß cells and elicits ß cell impairment.

Emerging evidence points toward an intricate relationship between the pandemic of coronavirus disease 2019 (COVID-19) and diabetes. While preexisting diabetes is associated with severe COVID-19, it is unclear whether COVID-19 severity is a cause or consequence of diabetes. To mechanistically link COVID-19 to diabetes, we tested whether insulin-producing pancreatic ß cells can be infected by SARS-CoV-2 and cause ß cell depletion. We found that the SARS-CoV-2 receptor, ACE2, and related entry factors (TMPRSS2, NRP1, and TRFC) are expressed in ß cells, with selectively high expression of NRP1. We discovered that SARS-CoV-2 infects human pancreatic ß cells in patients who succumbed to COVID-19 and selectively infects human islet ß cells in vitro. We demonstrated that SARS-CoV-2 infection attenuates pancreatic insulin levels and secretion and induces ß cell apoptosis, each rescued by NRP1 inhibition. Phosphoproteomic pathway analysis of infected islets indicates apoptotic ß cell signaling, similar to that observed in type 1 diabetes (T1D). In summary, our study shows SARS-CoV-2 can directly induce ß cell killing.

DMSO supplementation during in vitro maturation of bovine oocytes improves blastocyst rate and quality.

The molecule Dimethyl sulfoxide is widely used as drug solvent. However, its antioxidant property was poorly explored. In this study, we evaluated the effect of DMSO supplementation during oocyte in vitro maturation (IVM) on embryo development and quality. Bovine oocytes were matured with different DMSO concentrations (0, 0.1, 0.25, 0.5, 0.75, 1 and 10% v:v) followed by in vitro fertilization. Subsequently, quality indicators such as gene expression of SOX2, OCT4, CDX2, SOD1, oocyte and embryo redox status and DNA damage were evaluated. Polar body extrusion and blastocyst rates increased with 0.5% v:v DMSO. Moreover, first polar body extrusion and blastocyst rates did not increase with 1%, and 10% of DMSO reduced polar body extrusion and did not produce blastocyst. Optimal concentration of DMSO for the use on the maturation was estimated at around 0.45% v:v. Supplementation with 0.5% v:v DMSO has not affected mRNA abundance of genes key in blastocyst, however 0.75% increased gene expression of OCT4 and SOX2. Oocytes matured with 0.5% v:v DMSO and blastocyst from DMSO group showed reduced lipid peroxidation respect control. Total Glutathione concentrations increased in blastocyst stage in DMSO group. DMSO increased the total cell number of blastocysts but not TUNEL positive cells. In conclusion, our results suggest that low DMSO concentrations used during bovine oocytes in vitro maturation increases the maturation, as well as the blastocyst rate and its quality, without demonstrating deleterious effect on embryo cells.

Improved embryo development using high cysteamine concentration during IVM and sperm co-culture with COCs previous to ICSI in bovine.

In contrast to other species, intracytoplasmic sperm injection (ICSI) in bovine remains inefficient, resulting in low embryo developmental rates. It is unclear whether such inefficiency is due to the poor response of bovine ooplasms to the injection stimulus, or to the inability of bull sperm to induce oocyte activation. In order to facilitate these events, two strategies were assessed: the use of high concentration of cysteamine [Cys] during IVM; and the selection of sperm attached to cumulus cells after incubation with COCs for ICSI. First, COCs were IVM with increasing [Cys] and subjected to IVF. Zygotes from all groups were cultured under different O2 tensions and development to blastocyst was evaluated. In a second experiment, sperm were co-cultured for 3 h with COCs and acrosome reaction was studied. Afterwards, the best IVM and IVC conditions determined on Experiment 1 were used for ICSI assay. COCs were matured for 21 h with 1 (Cys 1) or 0.1 mM Cys (Cys 0.1 groups, standard condition). In addition, COCs were incubated for ≥3 h with 16 × 106 sperm/ml and only sperm attached to cumulus cells were selected for ICSI (ICSI + Co-cult groups). After chemical activation, embryos were cultured in SOF medium under low O2 tension. Cleavage and blastocyst rates were evaluated at days 2 and 7 of IVC, respectively. Finally, the relative expression of eight genes indicators of embryo quality was compared between ICSI and IVF control blastocysts by qPCR. Cleavage rates were higher for Cys 0.1 ICSI + Co-cult and Cys 1 ICSI + Co-cult groups (n = 117, 92% and n = 116, 79%, respectively) compared to their controls (n = 132, 60% for Cys 0.1 ICSI and n = 108, 52% for Cys 1 ICSI) (p ≤ 0.05). Interestingly, the combined treatment (Cys 1 ICSI + Co-cult) showed higher blastocyst rates than all other ICSI groups (23 vs. 11, 18 and 14% for Cys 0.1 ICSI + Co-cult, Cys 1 ICSI, and Cys 0.1 ICSI, respectively) (p ≤ 0.05). Moreover, incubation with COCs increased the rates of live acrosome reacted sperm (p ≤ 0.05). The relative abundance of mRNAs coding for INFτ, CAT, DNMT1, OCT4, and HDAC3 did not differ between treatments (p ≤ 0.05). SOD2, HADC1 and HADC2 expression was higher for Cys 0.1 ICSI than for IVF embryos (p ≤ 0.05). Group Cys 1 ICSI did not differ from IVF for those three genes, neither did Cys 1 ICSI + Co-cult, except for HDAC1 (p ≤ 0.05). In conclusion, the use of 1 mM Cys during IVM and of sperm incubated with mature COCs might be a good strategy to improve ICSI outcomes in cattle.

Sperm pretreatment with heparin and l-glutathione, sex-sorting, and double cryopreservation to improve intracytoplasmic sperm injection in bovine.

In bovine, intracytoplasmic sperm injection (ICSI) remains inefficient partially due to low levels of sperm decondensation. The aim of this study was to determine whether the injection of normal size sperm pretreated with heparin (Hep) and l-glutathione (GSH), the use of sex-sorted sperm, the double round of sperm freezing/thawing (re frozen), or the combination of these approaches can improve sperm decondensation and embryo development after ICSI in cattle. Cleavage and blastocyst rates were evaluated on days 2 and 7 post ICSI. Quality of ICSI blastocysts was analyzed by the relative expression of four genes by qPCR and the DNA fragmentation index by TUNEL assay. For all assays, semen samples were co-incubated with pCX-EGFP 50 ng/µl before ICSI. GFP expression, which can be detected by fluorescence microscopy, was used as a tool to estimate the success of sperm decondensation after ICSI. The use of normal size sperm pretreated with 80 µM Hep-15 mM GSH for 20 h (Hep-GSH) increased cleavage, blastocyst and EGFP + blastocysts rates (60.8, 19.4 and 61.9%) compared to control ICSI (35, 4.9 and 20%) (p < 0.05). Moreover, HMGN1, GLUT5, AQP3 and POU5F1 transcription levels did not differ between ICSI Hep-GSH and IVF embryos. The use of sex-sorted sperm (X, Y) improved cleavage rates and EGFP expression at day 4 (83 and 30.2% for ICSI Y and 83.2 and 31.7% for ICSI X) compared to non-sorted group (50.9 and 15.1%), not showing differences at the blastocyst stage. Finally, sex sorting (X) was combined with Hep-GSH and/or re frozen treatments. The use of Hep-GSH diminished cleavage rates from ICSI X re frozen group (80.4% vs. 94.2%) and blastocyst development from ICSI X group (3.3% vs. 10%), compared with their controls (p < 0.05). While Hep-GSH pretreatment induced lower transgene expression at day 4, no differences were found at the blastocyst stage between ICSI groups (from 58.3 to 80%). TUNEL assay showed higher DNA fragmentation indexes for all ICSI treatments (p < 0.05), except for ICSI X Hep-GSH, which did not differ from IVF X control. In conclusion, the use of normal size sperm pretreated with Hep-GSH, combined or not with sex-sorting by flow cytometry could improve ICSI outcomes in cattle.

Establishment of cell-based transposon-mediated transgenesis in cattle.

Transposon-mediated transgenesis is a well-established tool for genome modification in small animal models. However, translation of this active transgenic method to large animals warrants further investigations. Here, the piggyBac (PB) and sleeping beauty (SB) transposon systems were assessed for stable gene transfer into the cattle genome. Bovine fibroblasts were transfected either with a helper-independent PB system or a binary SB system. Both transposons were highly active in bovine cells increasing the efficiency of DNA integration up to 88 times over basal nonfacilitated integrations in a colony formation assay. SB transposase catalyzed multiplex transgene integrations in fibroblast cells transfected with the helper vector and two donor vectors carrying different transgenes (fluorophore and neomycin resistance). Stably transfected fibroblasts were used for SCNT and on in vitro embryo culture, morphologically normal blastocysts that expressed the fluorophore were obtained with both transposon systems. The data indicate that transposition is a feasible approach for genetic engineering in the cattle genome.