DCAF13 and RNF114 participate in the regulation of early porcine embryo development.

In brief: Ubiquitination plays a pivotal role in a multitude of cellular functions; however, the precise contributions of various ubiquitin ligases in governing early developmental processes remain largely unexplored. This study revealed that the E3 ubiquitin ligases DCAF13 and RNF114 are both necessary for the normal regulation of early porcine embryo development. Abstract: Ubiquitylation is required for normal regulation of many biological functions by modulating several protein facets such as structure, stability, interaction, localization, and degradation. In this study, we explored the roles of two E3 ubiquitin ligases (E3s), the DDB1- and CUL4-associated factor 13 (DCAF13) and the Ring finger protein 114 (RNF114), in the regulation of porcine embryo development. Attenuation of DCAF13 mRNA decreased embryo development at the blastocyst stage, while the development of RNF114-attenuated embryos was not significantly different than that of control embryos. The average number of cells per blastocyst was decreased in DCAF13-attenuated embryos and increased in RNF114-attenuated embryos compared to controls. The relative mRNA abundance of the histone methyltransferase SUV39H1, which regulates histone H3 lysine 9 trimethylation (H3K9me3), was increased in both DCAF13- and RNF114-attenuated embryos, but nuclear immunofluorescence signal for H3K9me3 on day 3 embryos was not significantly altered between attenuated and control embryos. Nuclear immunofluorescence signal for H3K4m3 was decreased in DCAF13-attenuated embryos, but it was increased in RNF114-attenuated embryos compared to controls. Attenuation of DCAF13 and RNF114 mRNAs increased transcript levels for the DNA recombinase RAD51 and decreased expression of phosphorylated histone H2A.X (γH2AX), which suggests an impact on DNA damage repair. In addition, lower mRNA expression of the lysine demethylases 5B (KDM5B) and 5C (KDM5C), both involved in embryo genome activation and DNA repair, was detected in DCAF13-attenuated embryos. These findings indicated that both DCAF13 and RNF114 have important roles in the regulation of the early development of porcine embryos.

Optimization of gonadotropin stimulation protocols for in vitro embryo production in prepubertal Mediterranean water buffalo.

Embryos can be produced from prepubertal donor animals using laparoscopic ovum pickup and in vitro embryo production technologies (LOPU-IVEP). Together, these tools can shorten the interval between generations, rapidly accelerating the rate of genetic gain. Here, we assessed the impact of different gonadotropin stimulation protocols in Mediterranean water buffalo heifer calves aged between 2 and 6 months old. Following gonadotropin stimulation, LOPU was performed at two-week intervals, with animals receiving different protocols on subsequent LOPUs. After collection, the cumulus-oocyte complexes (COCs) were matured and fertilized in vitro, and embryos were cultured to the blastocyst stage followed by transfer into synchronized adult recipients. The number and size of follicles aspirated during LOPU, the number and quality of COCs recovered, as well as cleavage, embryo development and pregnancy rates were assessed. First, we evaluated the impact of using FSH with and without eCG (administered 24-h prior to LOPU) and found that a combination of FSH and eCG was able to significantly improve embryo development rates (20.6 ± 2.0% vs. 9.0 ± 3.6%; P < 0.05). Second, we compared this protocol to a slow-release formulation of FSH reconstituted in hyaluronan. In addition to requiring less work to prepare the animals for LOPU, this slow-release formulation yielded numerically higher, but not statistically different, average number of recovered COCs (14.4 ± 2.1 vs. 10.3 ± 2.0; P > 0.05) and embryo development rates (22.9 ± 4.7% vs. 14.1 ± 5.2%; P > 0.05) compared to FSH given every 12 h. Next, we compared the length of gonadotropin treatment over 3-, 4- and 5-days prior to LOPU and found that as the length of gonadotropin treatment increased, although the number of COCs recovered steadily decreased (14.1 ± 2.4 vs. 8.7 ± 1.0 vs. 6.9 ± 0.7; P < 0.05), the embryo development rates steadily increased (14.4 ± 3.9 vs. 27.3 ± 4.4 vs. 35.9 ± 7.0; P < 0.05), presumably due to an increase in the proportion of large follicles at the time of LOPU. Numerically, the 4-day treatment yielded more transferrable embryos per donor per LOPU (2.70 ± 0.5) than 3-day (1.94 ± 0.6) and 5-day (2.25 ± 0.5) treatments. Finally, following embryo transfer, 26 of 90 recipient females became pregnant (28.9%). Pregnancies were established from all treatments, which suggests that post-implantation development was not affected among the gonadotropin treatments assessed.

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.

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.

FSH Regulates YAP-TEAD Transcriptional Activity in Bovine Granulosa Cells to Allow the Future Dominant Follicle to Exert Its Augmented Estrogenic Capacity.

The molecular mechanisms that drive the granulosa cells' (GC) differentiation into a more estrogenic phenotype during follicular divergence and establishment of follicle dominance have not been completely elucidated. The main Hippo signaling effector, YAP, has, however, emerged as a potential key player to explain such complex processes. Studies using rat and bovine GC demonstrate that, in conditions where the expression of the classic YAP-TEAD target gene tissue growth factor (CTGF) is augmented, CYP19A1 expression and activity and, consequently, estradiol (E2) secretion are reduced. These findings led us to hypothesize that, during ovarian follicular divergence in cattle, FSH downregulates YAP-TEAD-dependent transcriptional activity in GC to allow the future dominant follicle to exert its augmented estrogenic capacity. To address this, we performed a series of experiments employing distinct bovine models. Our in vitro and ex vivo experiments indicated that indeed FSH downregulates, in a concentration-dependent manner, mRNA levels not only for CTGF but also for the other classic YAP-TEAD transcriptional target genes ANKRD1 and CYR61 by a mechanism that involves increased YAP phosphorylation. To better elucidate the functional importance of such FSH-induced YAP activity regulation, we then cultured GC in the presence of verteporfin (VP) or peptide 17 (P17), two pharmacological inhibitors known to interfere with YAP binding to TEADs. The results showed that both VP and P17 increased CYP19A1 basal mRNA levels in a concentration-dependent manner. Most interestingly, by using GC samples obtained in vivo from dominant vs. subordinate follicles, we found that mRNA levels for CTGF, CYR61, and ANKRD1 are higher in subordinate follicles following the follicular divergence. Taken together, our novel results demonstrate that YAP transcriptional activity is regulated in bovine granulosa cells to allow the increased estrogenic capacity of the selected dominant follicle.

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.

The Hippo pathway effectors YAP and TAZ interact with EGF-like signaling to regulate expansion-related events in bovine cumulus cells in vitro.

PURPOSE: To determine if the inhibition of the interaction between the Hippo effector YAP or its transcriptional co-activator TAZ with the TEAD family of transcription factors is critical for the cumulus expansion-related events induced by the EGF network in cumulus-oocyte complexes (COCs). METHODS: We performed a series of experiments using immature bovine COCs subjected to an IVM protocol for up 24 h in which cumulus expansion was stimulated with EGF recombinant protein or FSH. RESULTS: The main results indicated that EGFR activity stimulation in bovine cumulus cells (CC) increases mRNA levels encoding the classic YAP/TAZ-TEAD target gene CTGF. To determine if important genes for cumulus expansion are transcriptional targets of YAP/TAZ-TEAD interaction in CC, COCs were then subjected to IVM in the presence of FSH with or without distinct concentrations of Verteporfin (VP; a small molecule inhibitor that interferes with YAP/TAZ binding to TEADs). COCs were then collected at 6, 12, 18, and 24 h for total RNA extraction and RT-qPCR analyses. This experiment indicated that VP inhibits in a time- and concentration-dependent manner distinct cumulus expansion and oocyte maturation-related genes, by regulating EGFR and CTGF expression in CC. CONCLUSIONS: Taken together, the results presented herein represent considerable insight into the functional relevance of a completely novel signaling pathway underlying cumulus expansion and oocyte maturation in monovulatory species. YAP/TAZ or CTGF may represent potential targets to improve the efficiency of IVM systems, not only for monovulatory species of agricultural importance as the cow, but for human embryo production.

Comparison between electroporation and polyfection in pig sperm: efficiency and cell viability implications.

SummaryThe aim of this study was to optimize protocols for electroporation (EP) and polyfection (PLF) using polyethyleneimine (PEI) for pig sperm transfection and to determine which method was the most efficient. For EP standardization, different voltages, amounts and times of electric pulses were tested using propidium iodide (PI) as reporter. For PLF standardization, different concentrations of fluorescein isothiocyanate (FITC)-labelled PEI (PEI/FITC) were incubated with sperm for different periods of time. Flow cytometry was performed to evaluate the best protocol in terms of cell viability, including cytoplasmic membrane, acrosome, chromatin integrities and mitochondrial potential using the FITC probe, PI, acridine orange (AO) and JC1. Transfections with the plasmid pmhyGENIE-5 were carried out under optimum conditions for each procedure (EP: 500 volts, 500 µs and two pulses; PLF: PEI 0.5 mg/ml and incubation time 10 min). Transfection efficacy was assessed by fluorescence in situ hybridization (FISH). A lower transfection rate was observed for sperm in the control group (17.8%) compared with EP (36.7%), with PLF (76.8%) being the most efficient. These results suggest that the EP and PEI could be an efficient and low cost transfection method for swine sperm. Notably, treated cells showed higher plasmatic the membrane damage (PMD) and/or acrosome damage (AD) indexes, therefore the combination of this procedure with biotechniques that facilitate fecundation (i.e. in vitro fertilization or intracytoplasmic sperm injection) or even inclusion of antioxidant or anti-apoptotic drugs to improve spermatozoa viability would be important.