Simultaneous Inhibition of Histone Deacetylases and RNA Synthesis Enables Totipotency Reprogramming in Pig SCNT Embryos

Combining somatic cell nuclear transfer (SCNT) with genome editing technologies has emerged as a powerful platform for the creation of unique swine lineages for agricultural and biomedical applications. However, successful application of this research platform is still hampered by the low efficiency of these technologies, particularly in attaining complete cell reprogramming for the production of cloned pigs. Treating SCNT embryos with histone deacetylase inhibitors (HDACis), such as Scriptaid, has been routinely used to facilitate chromatin reprogramming after nuclear transfer. While increasing histone acetylation leads to a more relaxed chromatin configuration that facilitates the access of reprogramming factors and DNA repair machinery, it may also promote the expression of genes that are unnecessary or detrimental for normal embryo development. In this study, we evaluated the impact of inhibiting both histone deacetylases and RNA synthesis on pre- and post-implantation development of pig SCNT embryos. Our findings revealed that transcription can be inhibited for up to 40 h of development in porcine embryos, produced either by activation, fertilization or SCNT, without detrimentally affecting their capacity to form a blastocyst and their average number of cells at this developmental stage. Importantly, inhibiting RNA synthesis during HDACi treatment resulted in SCNT blastocysts with a greater number of cells and more abundant transcripts for genes related to embryo genome activation on days 2, 3 and 4 of development, compared to SCNT embryos that were treated with HDACi only. In addition, concomitant inhibition of histone deacetylases and RNA synthesis promoted the full reprograming of somatic cells, as evidenced by the normal fetal and full-term development of SCNT embryos. This combined treatment may improve the efficiency of the genome-editing + SCNT platform in swine, which should be further tested by transferring more SCNT embryos and evaluating the health and growth performance of the cloned pigs

Modulation of physiological pathways to activate porcine oocytes

Oocyte activation is a physiological process triggered by the sperm during fertilization and is responsible for several events necessary for embryo development such as cortical granule exocytosis, elimination of the second polar body, cell cycle progression and pronuclear formation. Assisted oocyte activation is necessary for the production of porcine embryos through somatic cell nuclear transfer (SCNT), intracytoplasmic sperm injection (ICSI) and parthenogenetic activation. However, techniques such as SCNT and ICSI still show contradictory and low results. Although effective protocols for artificial oocyte activation have been developed, current protocols require long exposures to non-specific inhibitors, which do not mimic the physiological process and may have detrimental consequences for embryo development. In this research, we have explored physiological events induced following fertilization, through manipulation of calcium (Ca2+) and zinc (Zn2+) levels, and protein kinase C (PKC) and cyclin dependent kinase 1 (CDK1) activities, aiming at developing an improved protocol for activation of porcine oocytes. In the first experiment, matured oocytes were exposed to Ionomycin (Ion) for 5 min, and then treated with a specific CDK1 inhibitor (RO-3306) and/or PKC activator (OAG) for different times. The highest rate of pronuclear (PN) formation (58.8%) was obtained when oocytes were treated with CDK1i+PKCa for 4 h. Second, PN formation and embryo development were evaluated in oocytes exposed for different intervals to a Zn2+ chelator (TPEN) after Ion treatment. This revealed that 15 min was the minimal exposure time to TPEN required to maximize oocyte activation and embryo development. Next, we observed that treatment with CDK1i+PKCa for 4 h after TPEN for 15 min decreased embryo development compared with TPEN alone. Last, we compared the efficiency of the Ion (5 min) plus TPEN (15 min) protocol (IT-20) with a control protocol used in our laboratory (CT-245) for the production of PA, SCNT and ICSI embryos. In PA embryos, IT-20 resulted in higher cleavage (72% vs. 49.2%) and blastocyst from cleaved embryo (65.5% vs. 46.2%) rates compared with CT-245. In ICSI embryos, higher PN rates were obtained with the IT-20 protocol. Moreover, the two protocols were equally efficient for activation of SCNT embryos. Based on these findings, we propose that IT-20 is a faster and effective protocol for activation of porcine oocytes.L'activation de l'ovocyte est un processus physiologique déclenché par le spermatozoïde pendant la fécondation et elle est responsable pour plusieurs évènements nécessaires pour le développement de l'embryon, tel que l'exocytose des granules corticaux, l'expulsion du deuxième globule polaire, la progression du cycle cellulaire et la formation des pronoyaux (PN). Une activation assistée est nécessaire pour la production d'embryons porcins construits par transfert nucléaire de cellules somatiques (TNCS), injection intra-cytoplasmique de spermatozoïde (ICSI) et activation parthénogénétique (AP). Cependant, les méthodes telles que TNCS et ICSI démontrent toujours des résultats contradictoires et faibles. Bien que des protocoles efficaces pour l'activation artificielle d'ovocytes ont été développés, ces protocoles nécessitent une exposition prolongée à des inhibiteurs non-spécifiques, qui ne reproduit pas le processus physiologique, et peut même avoir des conséquences néfastes pour le développement de l'embryon. Cette recherche explore les évènements physiologiques provoqués suite à la fécondation, avec la manipulation des taux de Ca2+ et Zn2+, et l'activité des enzymes protéine kinase C (PKC) et kinase dépendante des cyclins 1 (CDK1), avec le but de développer un protocole amélioré pour l'activation des ovocytes porcins. Dans la première expérience, des ovocytes matures ont été exposés à ionomycine (Ion) pour 5 minutes, et ensuite traités avec un inhibiteur spécifique pour CDK1 (RO-3306) et/ou un activateur de PKC (OAG) pour différentes durées de temps. Le taux le plus élevé de formation de PN (58.8%) a été obtenu lorsque les ovocytes ont été traités avec CDK1i+PKCa pour 4 heures. Deuxièmement, la formation de PN et le développement embryonnaire ont été évalués lorsque des ovocytes ont été exposés, après traitement d'ionomycine, avec un chélateur de Zn2+ (TPEN) pour différentes périodes de temps. Ceci a révélé que 15 minutes était la durée minimale d'exposition à TPEN pour maximiser l'activation d'ovocytes et le développement embryonnaire. Ensuite, nous avons observé que le traitement avec CDK1i+PKCa pour 4 heures après TPEN pour 15 minutes a diminué le développement des embryons comparé avec seulement TPEN. Finalement, nous avons comparé l'efficacité du protocole d'Ion (5 min) suivi de TPEN (15 min) (IT-20) avec un protocole de contrôle utilisé dans notre laboratoire (CT-245) pour la production d'embryons par AP, TNCS et ICSI. IT-20 a augmenté le clivage (72% vs. 49.2%) et le taux de blastocyste selon clivage (65.5% vs. 46.2%) comparé à CT-245, dans les embryons développés par AP. Des taux plus élevés de formation PN ont été obtenu avec le protocole IT-20 lorsque les embryons ont été construits par ICSI. De plus, l'efficacité d'activation d'embryons construits par TNCS étaient pareilles avec les deux protocoles. Selon ces résultats, nous proposons que le protocole IT-20 est une méthode plus rapide et efficace pour l'activation des ovocytes porcins

NRF2 attenuation aggravates detrimental consequences of metabolic stress on cultured porcine parthenote embryos

Abstract The nuclear factor erythroid 2–related factor 2 (NRF2) is a crucial transcription factor that plays a central role in regulating oxidative stress pathways by binding antioxidant response elements, but its involvement in early embryo development remains largely unexplored. In this study, we demonstrated that NRF2 mRNA is expressed in porcine embryos from day 2 to day 7 of development, showing a decrease in abundance from day 2 to day 3, followed by an increase on day 5 and day 7. Comparable levels of NRF2 mRNA were observed between early-cleaving and more developmental competent embryos and late-cleaving and less developmental competent embryos on day 4 and day 5 of culture. Attenuation of NRF2 mRNA significantly decreased development of parthenote embryos to the blastocyst stage. When NRF2-attenuated embryos were cultured in presence of 3.5 mM or 7 mM glucose, development to the blastocyst stage was dramatically decreased in comparison to the control group (15.9% vs. 27.8% for 3.5 mM glucose, and 5.4% vs. 25.3% for 7 mM glucose). Supplementation of melatonin moderately improved the development of NRF2-attenuated embryos cultured in presence of 0.6 mM glucose. These findings highlight the importance of NRF2 in early embryo development, particularly in embryos cultured under metabolically stressful conditions

The histone lysine demethylase KDM7A is required for normal development and first cell lineage specification in porcine embryos

There is growing evidence that histone lysine demethylases (KDMs) play critical roles in the regulation of embryo development. This study investigated if KDM7A, a lysine demethylase known to act on mono-(me1) and di-(me2) methylation of H3K9 and H3K27, participates in the regulation of early embryo development. Knockdown of KDM7A mRNA reduced blastocyst formation by 69.2% in in vitro fertilized (IVF), 48.4% in parthenogenetically activated (PA), and 48.1% in somatic cell nuclear transfer (SCNT) embryos compared to controls. Global immunofluorescence (IF) signal in KDM7A knockdown compared to control embryos was increased for H3K27me1 on D7, for H3K27me2 on D3 and D5, for H3K9me1 on D5 and D7, and for H3K9me2 on D5 embryos, but decreased for H3K9me1, me2 and me3 on D3. Moreover, KDM7A knockdown altered mRNA expression, including the downregulation of KDM3C on D3, NANOG on D5 and D7, and OCT4 on D7 embryos, and the upregulation of CDX2, KDM4B and KDM6B on D5 embryos. On D3 and D5 embryos, total cell number and mRNA expression of embryo genome activation (EGA) markers (EIF1AX and PPP1R15B) were not affected by KDM7A knockdown. However, the ratio of inner cell mass (ICM)/total number of cells in D7 blastocysts was reduced by 45.5% in KDM7A knockdown compared to control embryos. These findings support a critical role for KDM7A in the regulation of early development and cell lineage specification in porcine embryos, which is likely mediated through the modulation of H3K9me1/me2 and H3K27me1/me2 levels, and changes in the expression of other KDMs and pluripotency genes

DNA Damage Induction Alters the Expression of Ubiquitin and SUMO Regulators in Preimplantation Stage Pig Embryos

DNA damage in early-stage embryos impacts development and is a risk factor for segregation of altered genomes. DNA damage response (DDR) encompasses a sophisticated network of proteins involved in sensing, signaling, and repairing damage. DDR is regulated by reversible post-translational modifications including acetylation, methylation, phosphorylation, ubiquitylation, and SUMOylation. While important regulators of these processes have been characterized in somatic cells, their roles in early-stage embryos remain broadly unknown. The objective of this study was to explore how ubiquitylation and SUMOylation are involved in the regulation of early development in porcine embryos by assessing the mRNA profile of genes encoding ubiquitination (UBs), deubiquitination (DUBs), SUMOylation (SUMOs) or deSUMOylation (deSUMOs) enzymes in oocyte and embryos at different stages of development, and to evaluate if the induction of DNA damage at different stages of embryo development would alter the mRNA abundance of these genes. Pig embryos were produced by in vitro fertilization and DNA damage was induced by ultraviolet (UV) light exposure for 10 s on days 2, 4 or 7 of development. The relative mRNA abundance of most UBs, DUBs, SUMOs, and deSUMOs was higher in oocytes and early-stage embryos than in blastocysts. Transcript levels for UBs (RNF20, RNF40, RNF114, RNF169, CUL5, DCAF2, DECAF13, and DDB1), DUBs (USP16), and SUMOs (CBX4, UBA2 and UBC9), were upregulated in early-stage embryos (D2 and/or D4) compared to oocytes and blastocysts. In response to UV-induced DNA damage, transcript levels of several UBs, DUBs, SUMOs, and deSUMOs decreased in D2 and D4 embryos, but increased in blastocysts. These findings revealed that transcript levels of genes encoding for important UBs, DUBs, SUMOs, and deSUMOs are regulated during early embryo development and are modulated in response to induced DNA damage. This study has also identified candidate genes controlling post-translational modifications that may have relevant roles in the regulation of normal embryo development, repair of damaged DNA, and preservation of genome stability in the pig embryo

Severe body condition loss lowers hepatic output of IGF1 with adverse effects on the dominant follicle in dairy cows

The severe loss of body condition score (BCS) during the early lactation period has been associated with infertility in cows. However, the mechanisms are not fully understood. The aim of this study was to examine the effect of BCS loss on liver health, and ovarian functions in cows during early lactation. Retrospectively multiparous cows from two farms were categorized based on units of BCS (1–5 scale) loss as Moderate (MOD, <0.75 units; n = 11) or Severe (SEV, ≥0.75 units; n = 9) loss groups. From Weeks −3 to 7, relative to calving, MOD and SEV cows lost on average 0.4 and 1.0-unit BCS, respectively. All data except hepatic transcriptomes were analyzed with PROC MIXED procedure of SAS. The plasma concentration of non-esterified fatty acids at Week 0 and 1, ß-hydroxy butyrate at Week 1, and γ-glutamyl transferase at Weeks 1 and 7 relative to calving were higher in SEV cows. Hepatic transcriptome analysis showed that 1 186 genes were differentially expressed in SEV (n = 3) compared to MOD (n = 3) cows at Week 7 after calving. Pathway analysis revealed that significant DEGs in SEV cows enriched in lipid metabolisms including, lipid metabolic process, ether lipid metabolism, fatty acid beta-oxidation, fatty acid biosynthetic process, fatty acid metabolic process, fat digestion and absorption, linoleic acid metabolism, alpha-linolenic acid metabolism. The impaired liver function in SEV cows was associated with 1.5-fold reduction of hepatic IGF1 gene expression and lower serum IGF1 concentrations. At the ovarian level, SEV cows had lower IGF1 concentration in the follicular fluid of the dominant follicle of the synchronized follicular wave compared to that of MOD cows at 7 weeks after calving. Further, the follicular fluid concentration of estradiol-17β was lower in SEV cows along with lower transcript abundance of genes from granulosa cells associated with dominant follicle competence, including CYP19A1, NR5A2, IGF1, and LHCGR. These data show that SEV loss of BCS during early lactation leading up to the planned start of breeding is associated with liver dysfunction, including lower IGF1 secretion, and impaired function of the dominant follicle in the ovary

Factors Affecting the Efficiency of In Vitro Embryo Production in Prepubertal Mediterranean Water Buffalo

Embryos from prepubertal water buffalo can be produced using laparoscopic ovum pickup (LOPU) and in vitro embryo production (IVEP). However, to date, it is unclear what factors and environmental conditions can affect LOPU-IVEP efficiency in prepubertal animals, especially buffalo. In this study, we explored the impact of season, age and individual variation among female donor animals, as well as the effect of the sire used for in vitro fertilization. Donor animals between 2 and 6 months of age were stimulated using gonadotropins prior to LOPU, which was performed at two-week intervals. Following in vitro maturation and fertilization, the resulting embryos were then cultured to the blastocyst stage until they were either vitrified or transferred into recipient animals. The number of follicles available for aspiration and embryo development rates was stable throughout the year. As animals became older, there was a slight trend for fewer COCs recovered from LOPU and better embryo development. There was a large individual variation in both ovarian response and the developmental competence of oocytes among donors. The bull used for fertilization also had a significant impact on embryo development. Upon embryo transfer, pregnancy rates were not affected by the number of embryos transferred per recipient. The best pregnancy rates were achieved when transferring blastocysts, compared to compact morula or hatched blastocysts. Finally, vitrification had no effect on pregnancy rate compared to fresh embryos