Granulocyte-colony stimulating factor (G-CSF), a pleiotropic cytokine, belongs to the hematopoietic growth factor family. Recent studies have reported that G-CSF is a predictive biomarker of oocyte ...and embryo developmental competence in humans. The aim of our study was to determine whether CSF3 and its receptor (CSF3R) were expressed in porcine maternal reproductive tissues (oviduct and uterus), cumulus cells, and embryos and to investigate the effects of human recombinant G-CSF (hrG-CSF) supplementation during in vitro culture (IVC) on the developmental competence of pre-implantation embryos. To do this, we first performed reverse-transcription polymerase chain reaction (RT-PCR). Second, we performed parthenogenetic activation (PA), in vitro fertilization (IVF), and somatic cell nuclear transfer (SCNT) to evaluate the embryonic developmental potential after hrG-CSF supplementation based on various concentrations (0 ng/mL, 10 ng/mL, 50 ng/mL, and 100 ng/mL) and durations (Un-treated, Days 0-3, Days 4-7, and Days 0-7) of IVC. Finally, we examined transcriptional levels of several marker genes in blastocysts. The results of our study showed that CSF3 transcript was present in all samples we assessed. CSF3-R was also detected, except in cumulus cells and blastocysts from PA. Furthermore, 10 ng/mL and Days 0-7 were the optimal concentration and duration for the viability of in vitro embryonic development, especially for SCNT-derived embryos. The rate of blastocyst formation and the total cell number of blastocysts were significantly enhanced, while the number and index of apoptotic nuclei were significantly decreased in optimal condition groups compared to others. Moreover, the transcriptional levels of anti-apoptotis- (BCL2), proliferation- (PCNA), and pluripotency- (POU5F1) related genes were dramatically upregulated. In conclusion, for the first time, we demonstrated that CSF3 and CSF3R were expressed in porcine reproductive organs, cells, and embryos. Additionally, we determined that hrG-CSF treatment improved porcine embryonic development capacity in vitro.
Granulocyte colony-stimulating factor (G-CSF), a pleiotropic cytokine, is secreted by the reproductive tract. Furthermore, our previous study indicated that human recombinant G-CSF (hrG-CSF) ...supplementation during porcine oocyte in vitro maturation (IVM) or during embryo in vitro culture (IVC) improved their quality and development potential when using cumulus-oocyte complexes (COCs) with more than three cumulus cell layers (CCL >3). Thus, in this study, we investigate the optimal conditions of hrG-CSF supplementation throughout the in vitro production (IVP: IVM + IVC) system to improve the embryo production efficiency of "poor-quality (CCL ≤3)" oocytes. COCs were classified into two groups according to the number of CCL (>3 and ≤3) and embryonic viability was analyzed after treatment with hrG-CSF during IVC. The mRNA transcription levels of G-CSF in COCs were compared based on their type and the period of IVM. Finally, developmental capacity and quality were evaluated after treatment with hrG-CSF for different periods of IVP. No marked effects on the developmental potential of embryos when using CCL ≤3 type COCs were observed after supplementing hrG-CSF only during IVC. Moreover, the mRNA transcription level of G-CSF increased gradually with IVM culture time and was higher in CCL ≤3 COCs than in >3. Supplementing hrG-CSF only during the IVM period resulted in the best embryo developmental potential, while supplementing hrG-CSF during the IVP period resulted in the best quality embryos, reflected in the increased total cell number and decreased apoptotic nuclei index of blastocysts. These findings indicate that "poor-quality" COCs may have a greater demand for G-CSF than "good-quality", meanwhile hrG-CSF supplementation throughout IVP improves resource utilization efficiency in poor-quality COCs.
Many studies have reported that interspecies somatic cell nuclear transfer (iSCNT) is considered the prominent method in preserving endangered animals. However, the development rate of iSCNT embryos ...is low, and there are limited studies on the molecular mechanism of the iSCNT process. This study evaluated the developmental potential of interspecies lycaon (Lycaon pictus)-dog embryos and assessed the mitochondrial content and metabolism of the produced cloned lycaon-dog fetus. Of 678 collected oocytes, 516 were subjected to nuclear transfer, and 419 reconstructed embryos with male lycaon fibroblasts were transferred into 27 surrogates. Of 720 oocytes, 568 were subjected to nuclear transfer and 469 reconstructed embryos with female lycaon fibroblasts were transferred into 31 surrogates. Two recipients who received female reconstructed embryos were identified as pregnant at 30 days. However, fetal retardation with no cardiac activity was observed at 46 days. Microsatellite analysis confirmed that the cloned lycaon-dog fetus was genetically identical to the lycaon donor cell, whereas mitochondrial sequencing analysis revealed that oocyte donor dogs transmitted their mtDNA. We assessed the oxygen consumption rate and mitochondrial content of the aborted lycaon-dog fetus to shed some light on the aborted fetus’s cellular metabolism. The oxygen consumption rates in the lycaon-dog fetal fibroblasts were lower than those in adult dog, lycaon and cloned dog fetal fibroblasts. Furthermore, lycaon-dog fetal fibroblasts showed decreased proportions of live and active mitochondria compared with other groups. Overall, we hypothesized that nuclear-mitochondrial incompatibility affects pyruvate metabolism and that these processes cause intrauterine fetal death.
•Lycaon embryos constructed with dog oocytes can implant in to the dog uterus.•Cloned lycaon-dog fetuses revealed lycaon nuclear DNA and heterogeneous mtDNA but, dominant dog mtDNA.•The OCR of lycaon fetal fibroblast was decreased compared to adult dog, lycaon and cloned dog fetal fibroblast.
Recent advances in somatic cell nuclear transfer (SCNT) in canines facilitate the production of canine transgenic models. Owing to the importance of stable and strong promoter activity in transgenic ...animals, we tested human elongation factor 1α (hEF1α) and cytomegalovirus (CMV) promoter sequences in SCNT transgenic dogs. After transfection, transgenic donor fibroblasts with the hEF1α-enhanced green fluorescence protein (EGFP) transgene were successfully isolated using fluorescence-activated cell sorting (FACS). We obtained four puppies, after SCNT, and identified three puppies as being transgenic using PCR analysis. Unexpectedly, EGFP regulated by hEF1α promoter was not observed at the organismal and cellular levels in these transgenic dogs. EGFP expression was rescued by the inhibition of DNA methyltransferases, implying that the hEF1α promoter is silenced by DNA methylation. Next, donor cells with CMV-EGFP transgene were successfully established and SCNT was performed. Three puppies of six born puppies were confirmed to be transgenic. Unlike hEF1α-regulated EGFP, CMV-regulated EGFP was strongly detectable at both the organismal and cellular levels in all transgenic dogs, even after 19 months. In conclusion, our study suggests that the CMV promoter is more suitable, than the hEF1α promoter, for stable transgene expression in SCNT-derived transgenic canine model.
The indiscriminate use of zinc oxide nanoparticles (ZnO NPs) in daily life can lead to their release into soil environment. These ZnO NPs can be taken up by crops and translocated to their edible ...part, potentially causing risks to the ecosystem and human health. In this study, we conducted pot experiments to determine phytotoxicity, bioaccumulation and translocation depending on the size (10 – 30 nm, 80 – 200 nm and 300 nm diameter) and concentration (0, 100, 500 and 1000 mg Zn/kg) of ZnO NPs and Zn ion (Zn2+) in bok choy, a leafy green vegetable crop. After 14 days of exposure, our results showed that large-sized ZnO NPs (i.e., 300 nm) at the highest concentration exhibited greater phytotoxicity, including obstruction of leaf and root weight (42.5 % and 33.8 %, respectively) and reduction of chlorophyll a and b content (50.2 % and 85.2 %, respectively), as well as changes in the activities of oxidative stress responses compared to those of small-sized ZnO NPs, although their translocation ability was relatively lower than that of smaller ones. The translocation factor (TF) values decreased as the size of ZnO NPs increased, with TF values of 0.68 for 10 – 30 nm, 0.55 for 80 – 200 nm, and 0.27 for 300 nm ZnO NPs, all at the highest exposure concentration. Both the results of micro X-ray fluorescence (μ-XRF) spectrometer and bio-transmission electron microscopy (bio-TEM) showed that the Zn elements were mainly localized at the edges of leaves exposed to small-sized ZnO NPs. However, the Zn elements upon exposure to large-sized ZnO NP were primarily observed in the primary veins of leaves in the μ-XRF data, indicating a limitation in their ability to translocate from roots to leaves. This study not only advances our comprehension of the environmental impact of nanotechnology but also holds considerable implications for the future of sustainable agriculture and food safety.
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•Size of ZnO NPs affects phytotoxicity and bioaccumulation/translocation in bok choy.•Smaller ZnO NPs show greater bioaccumulation and translocation abilities.•However, large-sized ZnO NPs (300 nm) exhibit higher phytotoxicity than smaller ones.•μ-XRF and bio-TEM provide information on the distribution of ZnO NPs within leaves.
Abstract
Background
This study aimed to evaluate cosmetic outcomes and feasibility of transvaginal natural orifice specimen extraction (NOSE) in patients who underwent laparoscopic or robotic surgery ...for the treatment of benign or malignant diseases of the kidney, liver, stomach, adrenal gland, and bladder.
Methods
This prospective study was conducted at a tertiary hospital between March 2015 and May 2020. The main outcome was cosmetic outcomes of scars assessed using the Patient and Observer Scar Assessment Scale (POSAS) 1 and 8 weeks after surgery. The secondary outcomes were postoperative pain, operating time, and complications. Sexual function was assessed using the Female Sexual Function Index (FSFI) questionnaire 6 months after surgery in 17 patients who were sexually active at the time of surgery.
Results
A total of 38 transvaginal NOSE procedures were performed for the extraction of 33 kidneys, 2 livers, 1 stomach, 1 adrenal gland, and 1 bladder. Observers rated pigmentation and relief scores as most deviant from normal skin (2.9 ± 1.7, 3.0 ± 2.1 at postoperative 1 week; 3.6 ± 1.9, 3.5 ± 2.2 at postoperative 8 weeks, respectively), but the overall scores of each item were low. The patients’ overall satisfaction with postoperative scars was high, and the mean scores for pain and itching were low, with significant improvement from the first week to the eighth week (
P
= 0.014 and
P
= 0.006, respectively). Patients also reported low scores on vaginal assessment items, indicating better symptoms, and bleeding improved significantly between the two time points (P = 0.001). Postoperative pain was reduced from moderate during the first 24 h after surgery to mild after 24 h. The mean operative time of the transvaginal NOSE procedure was 28.3 ± 13.3 min. No postoperative complications were associated with the procedure. The mean FSFI total score was 21.2 ± 8.7 (cutoff score for dysfunction is 21), with higher scores indicating better sexual functioning.
Conclusion
Transvaginal NOSE seems to be a feasible procedure with promising cosmetic benefits, for patients who undergo minimally invasive surgery for large organs including the kidney, liver, stomach, adrenal gland, and bladder. A prospective randomized clinical trial is needed to provide solid evidence to support transvaginal NOSE.
Trial registration:
This trial is registered at ClinicalTrials.gov (NCT05113134).
Several studies have reported methods to estimate the parturition date of dogs using ultrasonographic measurements. However, these prediction models were mainly determined using ultrasonographic ...measurements of naturally pregnant small- and medium-sized dogs, and no such studies have been performed using dogs carrying cloned fetuses produced via somatic cell nuclear transfer. The present study evaluated the abilities of three reference formulas (Luvoni and Grioni, Milani et al., and Groppetti et al.), all of which were developed using data from naturally occurring pregnancies, to accurately predict the parturition date in surrogates carrying cloned German Shepherd (GS) fetuses. All three formulas were based on the use of inner chorionic cavity diameter (ICC) measurements, obtained via ultrasonography. For evaluation, a total of 54 ICC measurements were collected from 14 pregnant bitches carrying cloned GS fetuses. We found that the clinical accuracy of the breed-specific Groppetti et al. formula was highest among those of the three formulas tested, with 87% and 100% of the estimated parturition dates (calculated based on the ICC measurements) being within 1 and 2 days, respectively, of the actual delivery date. By contrast, the Luvoni and Grioni formula showed relatively low accuracy, and the Milani et al. formula showed higher accuracy than that reported previously for natural pregnancies.
Although the etiology of Parkinson’s disease (PD) remains elusive, recent studies suggest that oxidative stress contributes to the cascade leading to dopaminergic (DAergic) neurodegeneration. The ...Nrf2 signaling is the main pathway responsible for cellular defense system against oxidative stress. Nrf2 is a transcription factor that regulates environmental stress response by inducing expression of antioxidant enzyme genes. We have synthesized novel vinyl sulfone derivatives. They exhibited a broad range of activities in inducing HO-1, whose gene expression is under the control of Nrf2. Among them, compound 12g was confirmed to activate Nrf2 and induce expression of the Nrf2-dependent antioxidant enzymes NQO1, GCLC, GLCM, and HO-1, at both mRNA and protein levels in DAergic neuronal cells. This was accompanied by protection of DAergic neurons in both in vitro and MPTP-induced in vivo models of PD. In addition, compound 12g effectively resulted in attenuation of the PD-associated behavioral deficits in the mouse model.
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