Application of Transposon Systems in The Transgenesis of Bovine Somatic And Germ Cells

Background: Several DNA transposons, PiggyBac (PB), Sleeping beauty (SB) and Tol2 have been applied as effective means for transgenesis in many species. Cattle are not typical experimental animals, and relatively little verification has been studied in this species. Thus, the goal of this study was the applicability of three transposon systems in somatic and embryo cells in cattle, while also determining which of the three systems is appropriate for each type of cell. To conduct the experiment, green fluorescent protein (GFP)-expressing transposon systems were used for electroporation and microinjection in the somatic cells and embryo stage, respectively. After transfection, GFP-positive cells or blastocysts were observed through a fluorescent microscope and transfection efficiency was calculated by FACS.Results: In the bovine somatic cells experiment, the PB (63.97 ± 11.56) showed higher efficiency as compared to the other two systems (SB: 50.74 ± 13.02 and Tol2: 16.55 ± 5.96). Unlike the results of the somatic cells, Tol2 (75.00%) and SB (70.00%) in the embryo were more efficient as compared to PB (42.86 %).Conclusions: These results demonstrate that all three transposon systems can be used in bovine somatic cells and embryos as a gene engineering experimental method and which type of transposon system is appropriate to apply depending on the cell type.

MiR-290 family maintains developmental potential by targeting p21 in mouse pre-implantation embryos

The miR-290 family is a mouse-specific microRNA cluster, which maintains mouse embryonic stem cells (ESCs) pluripotency by increasing OCT3/4 and C-MYC expression. However, its functions in mouse pre-implantation embryos remain unclear, especially during zygotic genome activation (ZGA). In this study, miR-290 family expression increased from the two-cell embryo stage through the blastocyst stage. Inhibition of miR-294-3p/5p did not affect ZGA initiation or embryo development, whereas pri-miR-290 knockdown decreased ZGA gene expression and slowed embryonic development. In addition, pluripotency decreased in ESCs derived from pri-miR-290 knockdown blastocysts. To clarify the mechanism of action, 33 candidate miR-294-3p target genes were screened from three databases, and miR-294-3p directly targeted the 3′-untranslated region of Cdkn1a (p21) mRNA. Similar to pri-miR-290 knockdown, P21 overexpression impeded embryonic development, whereas simultaneous overexpression of P21 and pri-miR-290 partially rescued embryonic development. The results indicate that the miR-290 family participates in promoting ZGA process and maintaining developmental potency in embryos by targeting p21.

Inhibition of DRP1 Impedes Zygotic Genome Activation and Preimplantation Development in Mice

Mitochondrion plays an indispensable role during preimplantation embryo development. Dynamic-related protein 1 (DRP1) is critical for mitochondrial fission and controls oocyte maturation. However, its role in preimplantation embryo development is still lacking. In this study, we demonstrate that inhibition of DRP1 activity by mitochondrial division inhibitor-1, a small molecule reported to specifically inhibit DRP1 activity, can cause severe developmental arrest of preimplantation embryos in a dose-dependent manner in mice. Meanwhile, DRP1 inhibition resulted in mitochondrial dysfunction including decreased mitochondrial activity, loss of mitochondrial membrane potential, reduced mitochondrial copy number and inadequate ATP by disrupting both expression and activity of DRP1 and mitochondrial complex assembly, leading to excessive ROS production, severe DNA damage and cell cycle arrest at 2-cell embryo stage. Furthermore, reduced transcriptional and translational activity and altered histone modifications in DRP1-inhibited embryos contributed to impeded zygotic genome activation, which prevented early embryos from efficient development beyond 2-cell embryo stage. These results show that DRP1 inhibition has potential cytotoxic effects on mammalian reproduction, and DRP1 inhibitor should be used with caution when it is applied to treat diseases. Additionally, this study improves our understanding of the crosstalk between mitochondrial metabolism and zygotic genome activation.

The comparative energetics of the turtles and crocodiles

The Add-my-Pet (AmP) collection of data on energetics and Dynamic Energy Budget (DEB) parameters currently contains 92 species of turtles and 23 species of crocodiles. We discuss patterns of eco-physiological traits of turtles and crocodiles, as functions of parameter values, and compare them with other taxa. Turtles and crocodiles accurately match the general rule that the life-time cumulated neonate mass production equals ultimate weight. The weight at birth for reptiles scales with ultimate weight to the power 0.6. The scaling exponent is between that of amphibians and birds, while that for mammals is close to 1. We explain why this points to limitations imposed by embryonic respiration, the role of water stress and the accumulation of nitrogen waste during the embryo stage. Weight at puberty is proportional to ultimate weight, and is the largest for crocodiles, followed by that of turtles. These facts explain why the precociality coefficient – approximated by the ratio of weight at birth and weight at puberty at abundant food – decreases with ultimate weight. It is the smallest for crocodile,s because of their large size, while that lizards and snakes are much larger than for turtles. The maximum reserve capacity in both turtles and crocodiles clearly decreases with the precociality coefficient. This relationship has not be found that clearly in other taxa, not even in other reptiles. Crocodiles have a relatively large assimilation rate and, as consequence, a large reserve capacity. Sea-turtles have a small weight and age at birth, which we link to reducing risks on the beach.

Mepanipyrim and cyprodinil induce divergent temporal patterns of AhR-mediated responses in zebrafish (Danio rerio) embryos and larvae

Mepanipyrim and cyprodinil are widely used to control and/or prevent fungal diseases in fruit culture. They are widely detected in aquatic environment and numerous food commodities including fruit and fruit products. Different from TCDD, mepanipyrim and cyprodinil are more easily degraded and metabolized in the environment. However, the in vivo analysis of their metabolic dynamics is unclear and need to be further confirmed. In this study, zebrafish embryos were constantly exposed to 100 µg/L mepanipyrim or cyprodinil for 7 days. The temporal pattern of CYP1A and AhR2 expression and EROD enzyme activity at different time frames during embryonic and larval development of zebrafish were investigated. Our results showed that mepanipyrim and cyprodinil tend to accumulate in zebrafish during early embryonic developmental stages. Meanwhile, mepanipyrim and cyprodinil exposure could increase the expression level of cyp1a and ahr2 genes and EROD activity by a dynamic pattern in different developmental stages of zebrafish. Besides, their metabolites, which may accumulate in the zebrafish larvae, have strong AhR agonistic activity and showed strong AhR binding ability. Importantly, the risk of exposure to pesticides in embryo stage is huge, and should be paid more attention.

Limits of Conventional Machine Learning Methods to Predict Pregnancy and Multiple Pregnancy After Embryo Transfer

When training models to learn the relationship between two or more variables, we expect to see previously demonstrated knowledge about that relationship reflected in the resulting estimators. For some domains, such as healthcare, it is imperative for actual implementation of those models that their predictions respect this knowledge. In this study we focus on Assisted Reproduction Technology (ART), the subspecialty of gynecology occupied with treating human infertility, and where the goal of any treatment is the delivery of a healthy newborn. A common ART treatment is In vitro Fertilization (IVF), where embryos are generated in vitro from collected sperm and oocytes, and transferred to the uterus of the patient after selecting those most likely to give rise to a healthy pregnancy. IVF has an approximate 30% successes rate per cycle; to palliate for this low success rate, a common practice so far has been to transfer two embryos simultaneously, aiming to increase the chances of a favorable outcome. While increasing overall live birth rates, this method has also led to an alarmingly high rate of twin and triplet births, associated with four times higher risk of perinatal mortality and increased obstetric complications. Our objective is to predict the chances of both pregnancy (P) and multiple pregnancy (MP) following either single embryo transfer (SET) or double embryo transfer (DET), and in so facilitating an informed decision on how many embryos to transfer. From existing literature, it is known that: (1) it is not possible for the chances of both P and MP to be decreased by increasing the number of embryos; (2) MP chances cannot be higher than P; and (3) chances of pregnancy are highly correlated with age, embryo stage, and quality. With a dataset generated from an existing observational study, we trained several state-of-the-art classifiers to predict P and MP given SET and DET. Analyzing the results, all classifiers achieved promising AUC scores. However, Random Forest and Gradient Boosting predicted negative chance differences in many instances when increasing the number of embryos infringing the first constraint. Logistic Regression predicted always positive differences, but in some instances it infringes the second constraint, predicting higher chances of MP than of P. Moreover, it showed little to no variation across ages or embryo stages violating third constraint. Conventional Machine Learning models struggle to reflect the real-world outcomes when using DET versus SET in specific patients. More informative variables could help, but it is already worrisome that variables as important as age and embryo stage do not result already in any variation, and that when models do show variation, in many cases they predicted decreasing chances of success with more embryos. We conclude that new and different approaches are needed to correctly model this scenario and, likely, many others resembling this one.

Reactive Oxygen Species Detection Using Fluorescence in Enchytraeus crypticus—Method Implementation through Ag NM300K Case Study

An imbalance between reactive oxygen species (ROS) and antioxidants in a living organism results in oxidative stress. Measures of such imbalance can be used as a biomarker of stress in ecotoxicology. In this study, we implemented the ROS detection method based on the oxidant-sensing probe dichloro-dihydro-fluorescein diacetate (DCFH-DA), detected by fluorescence microscopy, in Enchytraeus crypticus adults and cocoons, i.e., also covering the embryo stage. Hydrogen peroxide (H2O2), a well-known ROS inducer, was used both to optimize the method and as positive control. Implementation was successful, and the method was used to assess ROS formation in E. crypticus cocoons and adults when exposed to the reference silver nanomaterial Ag NM300K, at two effect concentrations (EC20 and EC50) for both hatching and reproduction over 3 and 7 days. The measured ROS levels varied with time, concentration, and developmental stage, with higher levels detected in adults compared with cocoons. In cocoons, ROS levels were higher at the EC20 than the EC50, which could be explained by non-monotonic concentration-response curve for hatching and reproduction, as previously observed. The increase in ROS levels at day 3 preceded the oxidative damage, as reported to occur later (day 7) in adults. The DCFH-DA method was successfully implemented here and can be further used as a new tool to detect ROS formation in E. crypticus, especially after short-term exposure to chemicals, including nanomaterials. We recommend the use of 3 and 7 days in the exposure design for this assessment.

Thin endometrium and cleavage embryo transfer are associated with ectopic pregnancy after in vitro fertilization‑embryo transfer cycles: a matched case–control study using propensity score matching

Objective: To evaluate the relationship between the endometrial features (endometrial thickness(EMT), pattern and endometrium growth amplitude and rate) and the embryo stage transferred and ectopic pregnancy after IVF-ET based on the data of 628 matched cases. Methods: This was a 1:1 matched case-control study that enrolled 314 EP patients and 314 matched IUP patients from the ART center of Xiangya Hospital of Central South University from January 2014 to April 2020. Univariate analysis and multiple-stepwise logistic regression analysis was used to analyze the risk factors of EP, and a receiver-operating characteristic (ROC) curve was generated to predict EP. Results: 1. The EP group had a higher cleavage stage embryo transfer rate(94.57% vs 86.22%), a thinner endometrium on transformation day (the day when progesterone was added in the frozen embryo cycle or the trigger day in the fresh embryo cycle) (9.40(2.60) mm vs 9.80(2.60)mm) compared with IUP group. In fresh embryo cycles, the EP group had a higher endometrium pattern C proportion on transformation day, a smaller endometrium increment, and a slower endometrial growth rate compared with the IUP group. 2.The ROC curves were used to analyze the cut-off values of the EMT on transformation day, the endometrial growth amplitude and rate in fresh cycles, the results were 9.35mm, 4.90mm and 0.491 mm/d, respectively. The incidence of EP was significantly different between groups according to cut-off values. 3. The transferred embryo stage and EMT on transformation day were independent factors affecting outcome in the general population. The area under the curve(AUC) of the EMT and the stage of embryos transferred for EP prediction was 0.604, sensitivity was 0.702, and specificity was 0.465. The EMT on trigger day was an independent factor affecting outcome in the fresh cycles. The AUC of the EMT for EP prediction in fresh cycles was 0.605, sensitivity was 0.805, and specificity was 0.420. Conclusions: 1. Transferring cleavage stage embryo , and thin EMT on the transformation day were risk factors for EP. 2. Thin EMT and pattern C on transformation day may be related to abnormal endometrial receptivity and endometrial peristaltic waves. 3.The combined indicator of EMT and embryonic development stage could not well predict the occurrence of EP after ET.

Comparative transcriptomic analyses of glucosinolate metabolic genes during the formation of Chinese kale seeds

Background To understand the mechanism of glucosinolates (GSs) accumulation in the specific organs, combined analysis of physiological change and transcriptome sequencing were applied in the current study. Taking Chinese kale as material, seeds and silique walls were divided into different stages based on the development of the embryo in seeds and then subjected to GS analysis and transcriptome sequencing. Results The main GS in seeds of Chinese kale were glucoiberin and gluconapin and their content changed with the development of the seed. During the transition of the embryo from torpedo- to the early cotyledonary-embryo stage, the accumulation of GS in the seed was accompanied by the salient decline of GS in the corresponding silique wall. Thus, the seed and corresponding silique wall at these two stages were subjected to transcriptomic sequencing analysis. 135 genes related to GS metabolism were identified, of which 24 genes were transcription factors, 81 genes were related to biosynthetic pathway, 25 genes encoded catabolic enzymes, and 5 genes matched with transporters. The expression of GS biosynthetic genes was detected both in seeds and silique walls. The high expression of FMOGS-OX and AOP2, which is related to the production of gluconapin by side modification, was noted in seeds at both stages. Interestingly, the expression of GS biosynthetic genes was higher in the silique wall compared with that in the seed albeit lower content of GS existed in the silique wall than in the seed. Combined with the higher expression of transporter genes GTRs in silique walls than in seeds, it was proposed that the transportation of GS from the silique wall to the seed is an important source for seed GS accumulation. In addition, genes related to GS degradation expressed abundantly in the seed at the early cotyledonary-embryo stage indicating its potential role in balancing seed GS content. Conclusions Two stages including the torpedo-embryo and the early cotyledonary-embryo stage were identified as crucial in GS accumulation during seed development. Moreover, we confirmed the transportation of GS from the silique wall to the seed and proposed possible sidechain modification of GS biosynthesis may exist during seed formation.