Involvement of METTL3-mediated m6A methylation in the early development of porcine cloned embryos.

METTL3-mediated N6-methyladenosine (m6A) modification is critical for gametogenesis and early embryonic development. However, the function of METTL3-mediated m6A modification in the early development of somatic nuclear transfer embryos (SCNT) remains unclear. Here, we found that METTL3 mRNA and protein levels exhibit dynamic changes during the early development of porcine SCNT embryos. The levels of METTL3 mRNA and protein in SCNT embryos at specific developmental stages differ from those in parthenogenetic activation (PA) counterparts. SiRNA injection effectively reduced the levels of METTL3 mRNA and protein in 4-cell embryos and blastocysts. METTL3 knockdown significantly reduced the cleavage and blastocyst rates of SCNT embryos. METTL3 knockdown significantly reduced the number of total cells and trophectoderm (TE) cells in the resulting blastocysts and perturbed cell lineage allocation. In addition, METTL3 knockdown reduced the levels of m6A modification in 4-cell embryos and blastocysts. Importantly, METTL3 knockdown decreased the expression levels of CDX2, GATA3, NANOG and YAP, and increased the expression levels of SOX2 and OCT4. Taken together, these results demonstrate that METTL3-mediated m6A modification regulates early development and lineage differentiation of porcine SCNT embryos.

Comparative transcriptome profile of embryos at different developmental stages derived from somatic cell nuclear transfer (SCNT) and in-vitro fertilization (IVF) in riverine buffalo (Bubalus bubalis).

Somatic cell nuclear transfer (SCNT) is a very important reproductive technology with many diverse applications, such as fast multiplication of elite animals, the production of transgenic animals and embryonic stem (ES) cells. However, low cloning efficiency, a low live birth rate and the abnormally high incidence of abnormalities in the offspring born are attributed to incomplete or aberrant nuclear reprogramming. In SCNT embryos, the aberrant expression pattern of the genes throughout embryonic development is responsible for the incomplete nuclear reprogramming. The present study was carried out to identify the differential gene expression (DEGs) profile and molecular pathways of the SCNT and IVF embryos at different developmental stages (2 cell, 8 cell and blastocyst stages). In the present study, 1164 (2 cell), 1004 (8 cell) and 530 (blastocyst stage) DEGs were identified in the SCNT embryos as compared to IVF embryos. In addition, several genes such as ZEB1, GDF1, HSF5, PDE3B, VIM, TNNC, HSD3B1, TAGLN, ITGA4 and AGMAT were affecting the development of SCNT embryos as compared to IVF embryos. Further, Gene Ontology (GO) and molecular pathways analysis suggested, SCNT embryos exhibit variations compared to their IVF counterparts and affected the development of embryos throughout the different developmental stages. Apart from this, q-PCR analysis of the GDF1, TMEM114, and IGSF22 genes were utilized to validate the RNA-seq data. These findings contribute valuable insights about the different genes and molecular pathways underlying SCNT embryo development and offer crucial information for improving SCNT efficiency.

Distinct properties of putative trophoblast stem cells established from somatic cell nuclear-transferred pig blastocysts.

BACKGROUND: Genetically modified pigs are considered ideal models for studying human diseases and potential sources for xenotransplantation research. However, the somatic cell nuclear transfer (SCNT) technique utilized to generate these cloned pig models has low efficiency, and fetal development is limited due to placental abnormalities. RESULTS: In this study, we unprecedentedly established putative porcine trophoblast stem cells (TSCs) using SCNT and in vitro-fertilized (IVF) blastocysts through the activation of Wing-less/Integrated (Wnt) and epidermal growth factor (EGF) pathways, inhibition of transforming growth factor-ß (TGFß) and Rho-associated protein kinase (ROCK) pathways, and supplementation with ascorbic acid. We also compared the transcripts of putative TSCs originating from SCNT and IVF embryos and their differentiated lineages. A total of 19 porcine TSCs exhibiting typical characteristics were established from SCNT and IVF blastocysts (TSCsNT and TSCsIVF). Compared with the TSCsIVF, TSCsNT showed distinct expression patterns suggesting unique TSCsNT characteristics, including decreased mRNA expression of genes related to apposition, steroid hormone biosynthesis, angiopoiesis, and RNA stability. CONCLUSION: This study provides valuable information and a powerful model for studying the abnormal development and dysfunction of trophoblasts and placentas in cloned pigs.

Essential roles of the nucleolus during early embryonic development: a regulatory hub for chromatin organization.

The nucleolus is the most prominent liquid droplet-like membrane-less organelle in mammalian cells. Unlike the nucleolus in terminally differentiated somatic cells, those in totipotent cells, such as murine zygotes or two-cell embryos, have a unique nucleolar structure known as nucleolus precursor bodies (NPBs). Previously, it was widely accepted that NPBs in zygotes are simply passive repositories of materials that will be gradually used to construct a fully functional nucleolus after zygotic genome activation (ZGA). However, recent research studies have challenged this simplistic view and demonstrated that functions of the NPBs go beyond ribosome biogenesis. In this review, we provide a snapshot of the functions of NPBs in zygotes and early two-cell embryos in mice. We propose that these membrane-less organelles function as a regulatory hub for chromatin organization. On the one hand, NPBs provide the structural platform for centric and pericentric chromatin remodelling. On the other hand, the dynamic changes in nucleolar structure control the release of the pioneer factors (i.e. double homeobox (Dux)). It appears that during transition from totipotency to pluripotency, decline of totipotency and initiation of fully functional nucleolus formation are not independent events but are interconnected. Consequently, it is reasonable to hypothesize that dissecting more unknown functions of NPBs may shed more light on the enigmas of early embryonic development and may ultimately provide novel approaches to improve reprogramming efficiency.

Reduction of H3K9 methylation by G9a inhibitors improves the development of mouse SCNT embryos.

Removal of somatic histone H3 lysine 9 trimethylation (H3K9me3) from the embryonic genome can improve the efficiency of mammalian cloning using somatic cell nuclear transfer (SCNT). However, this strategy involves the injection of histone demethylase mRNA into embryos, which is limiting because of its invasive and labor-consuming nature. Here, we report that treatment with an inhibitor of G9a (G9ai), the major histone methyltransferase that introduces H3K9me1/2 in mammals, greatly improved the development of mouse SCNT embryos. Intriguingly, G9ai caused an immediate reduction of H3K9me1/2, a secondary loss of H3K9me3 in SCNT embryos, and increased the birth rate of cloned pups about 5-fold (up to 3.9%). G9ai combined with the histone deacetylase inhibitor trichostatin A further improved this rate to 14.5%. Mechanistically, G9ai and TSA synergistically enhanced H3K9me3 demethylation and boosted zygotic genome activation. Thus, we established an easy, highly effective SCNT protocol that would enhance future cloning research and applications.

Impact of telomere length and mitochondrial DNA copy number variants on survival of newborn cloned calves.

An established technology to create cloned animals is through the use of somatic cell nuclear transfer (SCNT), in which reprogramming the somatic cell nucleus to a totipotent state by enucleated oocyte cytoplasm is a necessary process, including telomere length reprogramming. The limitation of this technology; however, is that the live birth rate of offspring produced through SCNT is significantly lower than that of IVF. Whether and how telomere length play a role in the development of cloned animals is not well understood. Only a few studies have evaluated this association in cloned mice, and fewer still in cloned cows. In this study, we investigated the difference in telomere length as well as the abundance of some selected molecules between newborn deceased cloned calves and normal cows of different ages either produced by SCNT or via natural conception, in order to evaluate the association between telomere length and abnormal development of cloned cows. The absolute telomere length and relative mitochondrial DNA (mtDNA) copy number were determined by real-time quantitative PCR (qPCR), telomere related gene abundance by reverse-transcription quantitative PCR (RT-qPCR), and senescence-associated ß-galactosidase (SA-ß-gal) expression by SA-ß-gal staining. The results demonstrate that the newborn deceased SCNT calves had significantly shortened telomere lengths compared to newborn naturally conceived calves and newborn normal SCNT calves. Significantly lower mtDNA copy number, and significantly lower relative abundance of LMNB1 and TERT, higher relative abundance of CDKN1A, and aberrant SA-ß-gal expression were observed in the newborn deceased SCNT calves, consistent with the change in telomere length. These results demonstrate that abnormal telomere shortening, lower mtDNA copy number and abnormal abundance of related genes were specific to newborn deceased SCNT calves, suggesting that abnormally short telomere length may be associated with abnormal development in the cloned calves.

Detection of newly synthesized RNA reveals transcriptional reprogramming during ZGA and a role of Obox3 in totipotency acquisition.

Zygotic genome activation (ZGA) after fertilization enables the maternal-to-zygotic transition. However, the global view of ZGA, particularly at initiation, is incompletely understood. Here, we develop a method to capture and sequence newly synthesized RNA in early mouse embryos, providing a view of transcriptional reprogramming during ZGA. Our data demonstrate that major ZGA gene activation begins earlier than previously thought. Furthermore, we identify a set of genes activated during minor ZGA, the promoters of which show enrichment of the Obox factor motif, and find that Obox3 or Obox5 overexpression in mouse embryonic stem cells activates ZGA genes. Notably, the expression of Obox factors is severely impaired in somatic cell nuclear transfer (SCNT) embryos, and restoration of Obox3 expression corrects the ZGA profile and greatly improves SCNT embryo development. Hence, our study reveals dynamic transcriptional reprogramming during ZGA and underscores the crucial role of Obox3 in facilitating totipotency acquisition.

Pronuclear transfer rescues poor embryo development of in vitro-grown secondary mouse follicles.

STUDY QUESTION: Is pronuclear transfer (PNT) capable of restoring embryo developmental arrest caused by cytoplasmic inferiority of in vitro-grown (IVG) mouse oocytes? SUMMARY ANSWER: PNT to in vivo matured cytoplasm significantly improved embryo development of IVG mouse oocytes, leading to living, fertile offspring. WHAT IS KNOWN ALREADY: In vitro follicle culture has been considered as a fertility preservation option for cancer patients. Studies describing the culture of human follicles remain scarce, owing to low availability of tissue. Mouse models have extensively been used to study and optimize follicle culture. Although important achievements have been accomplished, including the production of healthy offspring in mice, IVG oocytes are of inferior quality when compared to in vivo-grown oocytes, likely because of cytoplasmic incompetence. STUDY DESIGN SIZE DURATION: The study was carried out from September 2020 to February 2022. In total, 120 15-day-old B6D2 mice were used to perform secondary follicle culture and assess the quality of IVG oocytes. In vivo-grown control oocytes were obtained from 85 8- to 12-week-old B6D2 mice, following ovarian stimulation. For sperm collection, four B6D2 males between 10 and 14 weeks old were used. For embryo transfer, 14 8- to 12-week-old CD1 females served as surrogate mothers and 10 CD1 vasectomized males 10-24 weeks old were used to generate pseudo-pregnant females. Finally, for mating, four B6D2 female mice aged 8-10 weeks and two B6D2 male mice aged 10 weeks old were used to confirm the fertility of nuclear transfer (NT)-derived pups. PARTICIPANTS/MATERIALS SETTING METHODS: Secondary follicles from 15-day-old B6D2 mice were isolated from the ovaries and cultured for 9 days, before a maturation stimulus was given. Following 16-18 h of maturation, oocytes were collected and evaluated on maturation rate, oocyte diameter, activation rate, spindle morphology, calcium-releasing ability, and mitochondrial membrane potential. For every experiment, in vivo-grown oocytes were used as a control for comparison. When cytoplasmic immaturity and poor embryo development were confirmed in IVG oocytes, PNT was performed. For this, the pronuclei from IVG oocytes, created following parthenogenetic activation and IVF, were transferred to the cytoplasm of fertilized, in vivo-grown oocytes. Genetic analysis and embryo transfer of the generated embryos were implemented to confirm the safety of the technique. MAIN RESULTS AND THE ROLE OF CHANCE: Following 9 days of follicle culture, 703 oocytes were collected, of which 76% showed maturation to the metaphase II stage. Oocyte diameters were significantly lower in IVG oocytes, measuring 67.4 µm versus 73.1 µm in controls (P < 0.001). Spindle morphology did not differ significantly between IVG and control oocytes, but calcium-releasing ability was compromised in the IVG group. An average calcium release of 1.62 arbitrary units was observed in IVG oocytes, significantly lower than 5.74 in control oocytes (P < 0.001). Finally, mitochondrial membrane potential was inferior in IVG compared to the control group, reaching an average value of 0.95 versus 2.27 (P < 0.001). Developmental potential of IVG oocytes was assessed following parthenogenetic activation with strontium chloride (SrCl2). Only 59.4% of IVG oocytes cleaved to two cells and 36.3% reached the blastocyst stage, significantly lower than 89.5% and 88.2% in control oocytes, respectively (P < 0.001 and 0.001). Both PNT and spindle transfer (ST) were explored in pilot experiments with parthenogenetically activated oocytes, as a means to overcome poor embryo development. After the added value of NT was confirmed, we continued with the generation of biparental embryos by PNT. For this purpose, IVG and control oocytes first underwent IVF. Only 15.5% of IVG oocytes were normally fertilized, in contrast to 45.5% in controls (P < 0.001), with resulting failure of blastocyst formation in the IVG group (0 versus 86.2%, P < 0.001). When the pronuclei of IVG zygotes were transferred to the cytoplasm of control zygotes, the blastocyst rate was restored to 86.9%, a similar level as the control. Genetic analysis of PNT embryos revealed a normal chromosomal profile, to a rate of 80%. Finally, the generation of living, fertile offspring from PNT was possible following embryo transfer to surrogate mothers. LARGE-SCALE DATA: N/A. LIMITATIONS REASONS FOR CAUTION: Genetic profiles of analysed embryos from PNT originate from groups that are too small to draw concrete conclusions, whilst ST, which would be the preferred NT approach, could not be used for the generation of biparental embryos owing to technical limitations. Even though promising, the use of PNT should be considered as experimental. Furthermore, results were acquired in a mouse model, so validation of the technique in human IVG oocytes needs to be performed to evaluate the clinical relevance of the technology. The genetic profiles from IVG oocytes, which would be the ultimate characterization for chromosomal abnormalities, were not analysed owing to limitations in the reliable analysis of single cells. WIDER IMPLICATIONS OF THE FINDINGS: PNT has the ability to overcome the poor cytoplasmic quality of IVG mouse oocytes. Considering the low maturation efficiency of human IVG oocytes and potential detrimental effects following long-term in vitro culture, NT could be applied to rescue embryo development and could lead to an increased availability of good quality embryos for transfer. STUDY FUNDING/COMPETING INTERESTS: A.C. is a holder of FWO (Fonds voor Wetenschappelijk Onderzoek) grants (1S80220N and 1S80222N). B.H. and A.V.S. have been awarded with a special BOF (Bijzonder Onderzoeksfonds), GOA (Geconcerteerde onderzoeksacties) 2018000504 (GOA030-18 BOF) funding. B.H. has been receiving unrestricted educational funding from Ferring Pharmaceuticals (Aalst, Belgium). The authors declare that they have no conflict of interest.

Outlook on genome editing application to cattle.

In livestock industry, there is growing interest in methods to increase the production efficiency of livestock to address food shortages, given the increasing global population. With the advancements in gene engineering technology, it is a valuable tool and has been intensively utilized in research specifically focused on human disease. In historically, this technology has been used with livestock to create human disease models or to produce recombinant proteins from their byproducts. However, in recent years, utilizing gene editing technology, cattle with identified genes related to productivity can be edited, thereby enhancing productivity in response to climate change or specific disease instead of producing recombinant proteins. Furthermore, with the advancement in the efficiency of gene editing, it has become possible to edit multiple genes simultaneously. This cattle breed improvement has been achieved by discovering the genes through the comprehensive analysis of the entire genome of cattle. The cattle industry has been able to address gene bottlenecks that were previously impossible through conventional breeding systems. This review concludes that gene editing is necessary to expand the cattle industry, improving productivity in the future. Additionally, the enhancement of cattle through gene editing is expected to contribute to addressing environmental challenges associated with the cattle industry. Further research and development in gene editing, coupled with genomic analysis technologies, will significantly contribute to solving issues that conventional breeding systems have not been able to address.

ACADL-YAP axis activity in non-small cell lung cancer carcinogenicity.

BACKGROUND: The role of Acyl-CoA dehydrogenase long chain (ACADL) in different tumor types had different inhibiting or promoting effect. However, its role in non-small cell lung cancer (NSCLC) carcinogenicity is not clear. METHOD: In this study, we utilized The Cancer Genome Atlas (TCGA) database to analyze ACADL expression in NSCLC and its correlation with overall survival. Furthermore, we investigated the function of ACADL on cellular proliferation, invasion, colony, apoptosis, cell cycle in vitro with NSCLC cells. Mechanistically, we evaluated the regulatory effect of ACADL expression on its downstream factor yes-associated protein (YAP) by assessing YAP phosphorylation levels and its cellular localization. Finally, we verified the tumorigenic effect of ACADL on NSCLC cells through xenograft experiments in vivo. RESULTS: Compared to adjacent non-cancerous samples, ACADL significantly down-regulated in NSCLC. Overexpression of ACADL, effectively reduced the proliferative, colony, and invasive capabilities of NSCLC cells, while promoting apoptosis and inducing cell cycle arrest. Moreover, ACADL overexpression significantly enhanced YAP phosphorylation and hindered its nuclear translocation. However, the inhibitory effect of the overexpression of ACADL in NSCLC cells mentioned above can be partially counteracted by YAP activator XMU-MP-1 application both in vitro and in vivo. CONCLUSION: The findings suggest that ACADL overexpression could suppress NSCLC development by modulating YAP phosphorylation and limiting its nuclear shift. This role of ACADL-YAP axis provided novel insights into NSCLC carcinogenicity and potential therapeutic strategies.

Analysis of production efficiency of cloned transgenic Yucatan miniature pigs according to recipient breeds with embryo transfer conditions.

The purpose of this study was to compare the efficiency of the production of cloned transgenic Yucatan miniature pigs (YMPs) using two recipient breeds, i.e., YMPs and domestic pigs (DPs), under various embryo transfer conditions. We initially assessed the in vitro developmental competence of embryos obtained via somatic cell nuclear transfer (SCNT) from three different transgenic donor cells. No difference was observed among the three groups regarding developmental competence. Furthermore, the cloning efficiency remained consistent among the three groups after the transfer of the SCNT embryos to each surrogate mother. Subsequently, to compare the efficiency of the production of cloned transgenic YMPs between the two recipient breeds using varying parameters, including ovulation status (preovulation and postovulation), duration of in vitro culture (IVC) (incubated within 24 h and 24-48 h), and the number of transferred SCNT embryos (less than and more than 300), we assessed the pregnancy rates, delivery rates, mean offspring counts, and cloning efficiency. Regarding the ovulation status, YMPs exhibited higher pregnancy rates, delivery rates, and cloning efficiency compared with DPs in both statuses. Moreover, the pregnancy rates, delivery rates, and cloning efficiency were affected by the ovulation status in DPs, but not in YMPs. The comparison of IVC duration between groups revealed that YMPs had higher pregnancy rates vs. DPs in both conditions. SCNT embryos cultured for 24-48 h in YMPs yielded higher delivery rates and cloning efficiency compared with those cultured for less than 24 h in DPs. Finally, the analysis based on the number of transferred SCNT embryos showed that both the pregnancy and delivery rates were higher in YMPs vs. DPs. However, the highest average number of offspring was obtained when more than 300 SCNT embryos were transferred into DPs, whereas the cloning efficiency was higher in YMPs vs. DPs. Our results suggest that YMPs are more suitable recipients than are DPs under various conditions for the production of cloned transgenic YMPs.

Cloned Foal Born from Postmortem-Obtained Ear Sample Refrigerated for 5 Days Before Fibroblast Isolation and Decontamination of the Infected Monolayer Culture.

A 6-year-old mare, a valuable polo horse, died of complications following postcolic surgery. To preserve its genetics, ear skin samples were collected immediately after death and stored in an equine embryo transfer medium at 4°C for 5 days. After trypsin digestion, monolayer fibroblast cultures were established, but signs of massive bacterial infection were found in all of them. As an ultimate attempt for rescue, rigorously and repeatedly washed cells were individually cultured in all wells of four 96-well dishes. New monolayers were established from the few wells without contamination and used for somatic cell nuclear transfer. Four of the six Day 7 blastocysts derived from 14 reconstructed zygotes were transferred in four naturally cycling mares on Day 5 after ovulation. The embryo transfers resulted in 2 pregnancies, one from a fresh and one from a vitrified blastocyst. The vitrified embryo transfer resulted in a healthy offspring, now 21 months old, genetically and phenotypically identical to the somatic cell donor animal.

Incomplete reprogramming of DNA replication timing in induced pluripotent stem cells.

Induced pluripotent stem cells (iPSCs) are the foundation of cell therapy. Differences in gene expression, DNA methylation, and chromatin conformation, which could affect differentiation capacity, have been identified between iPSCs and embryonic stem cells (ESCs). Less is known about whether DNA replication timing, a process linked to both genome regulation and genome stability, is efficiently reprogrammed to the embryonic state. To answer this, we compare genome-wide replication timing between ESCs, iPSCs, and cells reprogrammed by somatic cell nuclear transfer (NT-ESCs). While NT-ESCs replicate their DNA in a manner indistinguishable from ESCs, a subset of iPSCs exhibits delayed replication at heterochromatic regions containing genes downregulated in iPSCs with incompletely reprogrammed DNA methylation. DNA replication delays are not the result of gene expression or DNA methylation aberrations and persist after cells differentiate to neuronal precursors. Thus, DNA replication timing can be resistant to reprogramming and influence the quality of iPSCs.

Aggregated chromosomes/chromatin transfer: a novel approach for mitochondrial replacement with minimal mitochondrial carryover: the implications of mouse experiments for human aggregated chromosome transfer.

Nuclear transfer techniques, including spindle chromosome complex (SC) transfer and pronuclear transfer, have been employed to mitigate mitochondrial diseases. Nevertheless, the challenge of mitochondrial DNA (mtDNA) carryover remains unresolved. Previously, we introduced a method for aggregated chromosome (AC) transfer in human subjects, offering a potential solution. However, the subsequent rates of embryonic development have remained unexplored owing to legal limitations in Japan, and animal studies have been hindered by a lack of AC formation in other species. Building upon our success in generating ACs within mouse oocytes via utilization of the phosphodiesterase inhibitor 3-isobutyl 1-methylxanthine (IBMX), this study has established a mouse model for AC transfer. Subsequently, a comparative analysis of embryo development rates and mtDNA carryover between AC transfer and SC transfer was conducted. Additionally, the mitochondrial distribution around SC and AC structures was investigated, revealing that in oocytes at the metaphase II stage, the mitochondria exhibited a relatively concentrated arrangement around the spindle apparatus, while the distribution of mitochondria in AC-formed oocytes appeared to be independent of the AC position. The AC transfer approach produced a marked augmentation in rates of fertilization, embryo cleavage, and blastocyst formation, especially as compared to scenarios without AC transfer in IBMX-treated AC-formed oocytes. No significant disparities in fertilization and embryo development rates were observed between AC and SC transfers. However, relative real-time PCR analyses revealed that the mtDNA carryover for AC transfers was one-tenth and therefore significantly lower than that of SC transfers. This study successfully accomplished nuclear transfers with ACs in mouse oocytes, offering an insight into the potential of AC transfers as a solution to heteroplasmy-related challenges. These findings are promising in terms of future investigation with human oocytes, thus advancing AC transfer as an innovative approach in the field of human nuclear transfer methodology.

Quantitative analysis of mitochondrial DNA in porcine-mouse cloned embryos.

The aim of the research is to identify that porcine oocytes can function as recipients for interspecies cloning and have the ability to develop to blastocysts. Furthermore each mitochondrial DNA (mtDNA) in interspecises cloned embryos was analyzed. For the study, mouse-porcine and porcine-porcine cloned embryos were produced with mouse fetal fibroblasts (MFF) and porcine fetal fibroblasts (PFF), respectively, introduced as donor cells into enucleated porcine oocytes. The developmental rate and cell numbers of blastocysts between intraspecies porcine-porcine and interspecies mouse-porcine cloned embryos were compared and real-time polymerase chain reaction (PCR) was performed for the estimate of mouse and porcine mtDNA copy number in mouse-porcine cloned embryos at different stages.There was no significant difference in the developmental rate or total blastocyst number between mouse-porcine cloned embryos and porcine-porcine cloned embryos (11.1 ± 0.9%, 25 ± 3.5 vs. 10.1 ± 1.2%, 24 ± 6.3). In mouse-porcine reconstructed embryos, the copy numbers of mouse somatic cell-derived mtDNA decreased between the 1-cell and blastocyst stages, whereas the copy number of porcine oocyte-derived mtDNA significantly increased during this period, as assessed by real-time PCR analysis. In our real-time PCR analysis, we improved the standard curve construction-based method to analyze the level of mtDNA between mouse donor cells and porcine oocytes using the copy number of mouse beta-actin DNA as a standard. Our findings suggest that mouse-porcine cloned embryos have the ability to develop to blastocysts in vitro and exhibit mitochondrial heteroplasmy from the 1-cell to blastocyst stages and the mouse-derived mitochondria can be gradually replaced with those of the porcine oocyte in the early developmental stages of mouse-porcine cloned embryos.

Targeting NF-κB in Hepatic Ischemia-Reperfusion Alleviation: from Signaling Networks to Therapeutic Targeting.

Hepatic ischemia-reperfusion injury (HIRI) is a major complication of liver trauma, resection, and transplantation that can lead to liver dysfunction and failure. Scholars have proposed a variety of liver protection methods aimed at reducing ischemia-reperfusion damage, but there is still a lack of effective treatment methods, which urgently needs to find new effective treatment methods for patients. Many studies have reported that signaling pathway plays a key role in HIRI pathological process and liver function recovery mechanism, among which nuclear transfer factor-κB (NF-κB) signaling pathway is one of the signal transduction closely related to disease. NF-κB pathway is closely related to HIRI pathologic process, and inhibition of this pathway can delay oxidative stress, inflammatory response, cell death, and mitochondrial dysfunction. In addition, NF-κB can also interact with PI3K/Akt, MAPK, and Nrf2 signaling pathways to participate in HIRI regulation. Based on the role of NF-κB pathway in HIRI, it may be a potential target pathway for HIRI. This review emphasizes the role of inhibiting the NF-κB signaling pathway in oxidative stress, inflammatory response, cell death, and mitochondrial dysfunction in HIRI, as well as the effects of related drugs or inhibitors targeting NF-κB on HIRI. The objective of this review is to elucidate the role and mechanism of NF-κB pathway in HIRI, emphasize the important role of NF-κB pathway in the prevention and treatment of HIRI, and provide a theoretical basis for the target NF-κB pathway as a therapy for HIRI.

Effects of alpha lipoic acid treatment during in vitro maturation on the development of porcine somatic cell nuclear transfer embryos.

Oxidative stress influences the embryo production efficiency in vitro. We investigated the effects of alpha lipoic acid (ALA) treatment during the in vitro maturation (IVM) period on the porcine somatic cell nuclear transfer (SCNT) embryo production. After IVM, maturation rates of the 12.5- and 25-µM ALA-treated groups were not significantly different from those of the 0-µM ALA-treated group. Compared to those in the 0-µM ALA-treated group, the reactive oxygen species and glutathione levels were significantly decreased and increased, respectively, in the cytoplasm of matured oocytes in the 12.5-50-µM ALA-treated groups. Apoptosis rate in cumulus cells after IVM was significantly lower in the 12.5-50-µM ALA-treated groups than in the 0-µM ALA-treated group. Blastocyst formation rate was significantly higher in parthenogenetic oocytes treated with 12.5-µM ALA than in the 0-, 25-, and 50-µM ALA-treated groups. Similarly, in SCNT embryos, the 12.5-µM ALA-treated group showed a significantly higher blastocyst formation rate than the 0-µM ALA-treated group. Apoptosis rate in SCNT blastocysts was significantly decreased by 12.5-µM ALA treatment. The results showed that treatment with 12.5-µM ALA during IVM improves porcine SCNT embryo development and partial quality.

Stem cell factor's role in enhancing the quality of fertilized and cloned porcine embryos for improved embryonic stem cell derivation.

Stem cell factor (SCF), a cytokine growth factor, is expressed in various tissues of the male and female reproductive organs, including the testis, ovary, and endometrium. Its primary function involves cell survival, differentiation, and proliferation, achieved through its binding to the c-kit receptor. This study aimed to scrutinize the effects of SCF treatment during in vitro culture (IVC) on both the developmental potential and the efficiency of establishing embryonic stem cells (ESCs) from fertilized and cloned porcine embryos. The rates of cleavage and blastocyst formation exhibited no significant differences between fertilized and cloned embryos, even with the addition of SCF. However, it's worth noting that embryos cloned with Cloud eGFP as donor cells demonstrated notably increased rates of hatched blastocysts when treated with SCF, and this increase was statistically significant (p < 0.05). Furthermore, following the complete dissection of the blastocysts, although there was no significant difference in the SCF-treated group, the area of expansion was significantly reduced (p < 0.01) in the group treated with the antagonistic blocker (ACK2) compared to both the control and SCF-treated groups. These outcomes suggest that the SCF/c-kit signaling pathway might play a pivotal role in embryo implantation. As anticipated, the efficiency of deriving ESCs was significantly higher (p < 0.01) in the group subjected to SCF treatment (12.82 ± 1.02%) compared to the control group (5.41 ± 2.25%). In conclusion, this study highlights the crucial role of SCF in enhancing the quality of porcine embryos, a vital step in obtaining high-quality ESCs.

Complex haploinsufficiency in pluripotent cells yields somatic cells with DNA methylation abnormalities and pluripotency induction defects.

A complete knockout of a single key pluripotency gene may drastically affect embryonic stem cell function and epigenetic reprogramming. In contrast, elimination of only one allele of a single pluripotency gene is mostly considered harmless to the cell. To understand whether complex haploinsufficiency exists in pluripotent cells, we simultaneously eliminated a single allele in different combinations of two pluripotency genes (i.e., Nanog+/-;Sall4+/-, Nanog+/-;Utf1+/-, Nanog+/-;Esrrb+/- and Sox2+/-;Sall4+/-). Although these double heterozygous mutant lines similarly contribute to chimeras, fibroblasts derived from these systems show a significant decrease in their ability to induce pluripotency. Tracing the stochastic expression of Sall4 and Nanog at early phases of reprogramming could not explain the seen delay or blockage. Further exploration identifies abnormal methylation around pluripotent and developmental genes in the double heterozygous mutant fibroblasts, which could be rescued by hypomethylating agent or high OSKM levels. This study emphasizes the importance of maintaining two intact alleles for pluripotency induction.

Reproduction of Sheep through Nuclear Transfer of Somatic Cells: A Bibliometric Approach.

Somatic cell nuclear transfer (SCNT) is a reproductive biotechnology with great potential in the reproduction of different species of zootechnical interest, including sheep. This study aimed to carry out a bibliometric analysis of scientific papers published on the application of SCNT in sheep reproduction during the period 1997-2023. The search involved the Science Citation Index Expanded and Social Sciences Citation Index databases of the main collection of the Web of Sciences with different descriptors. A total of 124 scientific papers were analyzed for different bibliometric indicators using the VOSviewer software. Since 2001, the number of SCNT-related papers that have been published concerning sheep reproduction has increased and it has fluctuated in ensuing years. The main authors, research groups, institutions, countries, papers, and journals with the highest number of papers related to the application of SCNT in sheep reproduction were identified, as well as the topics that address the research papers according to the terms: somatic cell, embryo, oocyte, gene expression, SCNT, and sheep.