Assessing the efficacy of mesotherapy products: Ultra Exo Booster, and Ultra S Line Plus in hair growth: An ex vivo study.

In this study, scalp tissues from Korean adults between 20 and 80 without skin disease were used. Scalp tissues were processed, and hair follicles were isolated and cultured with different treatments (including Bioscalp, Ultra Exo Booster, and Ultra S Line Plus) from Ultra V company. Over 12 days, observations and measurements of hair follicle characteristics were recorded at intervals (Days 0, 3, 6, 9, and 12). The study assessed the impact of these substances on hair follicle growth and morphology. Bioscalp, combined with Ultra Exo Booster and Ultra S Line Plus, showed significant hair elongation in ex vivo. Preservation of hair bulb diameter was observed, indicating potential for sustained hair growth by exosome-based products. The hair growth cycle analysis suggested a lower transition to the catagen stage in test products from Ultra V compared to non-treated groups. The research findings indicated that the tested formulations, especially the combination of Bioscalp, Ultra Exo Booster, and Ultra S Line Plus, demonstrated significant effectiveness in promoting hair growth, maintaining the integrity of the hair bulb, and reducing the transition to the catagen stage. The study suggests promising alternative treatments for hair loss, illustrating results that were as good as those of the conventional testing product groups.

Evaluation test and analysis of a microneedle and iontophoresis based medical device "CELLADEEP Patch" in skin improvement on ex vivo human-derived skin tissue models.

BACKGROUND: Microneedles are tiny needles, typically ranging from tens to hundreds of micrometers in length, used in various medical procedures and treatments. The tested medical device named "CELLADEEP Patch" a dissolvable microneedle therapy system (MTS), made of hyaluronic acid and collagen. And the iontophoresis technique is also applied in the system. The study aimed to evaluate the effectiveness of the "CELLADEEP Patch" in skin improvement. METHODS: Ex vivo human-derived skin tissue models were used in this study and they were divided into three different groups, namely, the Untreated Group, the Negative Control Group, and the Test Group respectively. The Untreated Group received no treatment measures, the Negative Control Group was exposed to ultraviolet B radiation (UVB) irradiation, and the Test Group was exposed to UVB irradiation and treated with "CELLADEEP Patch". Skin moisture content, transdermal water loss, and skin elasticity were evaluated by three clinical devices. Additionally, histological staining and related mRNA expression levels were also analyzed. RESULTS: The results of skin moisture content, transdermal water loss, and skin elasticity evaluation consistently illustrated that the application of "CELLADEEP Patch" led to remarkable skin improvement. And the analysis of histological staining images also confirmed the effectiveness of the "CELLADEEP Patch", especially for increasing collagen density. Moreover, the upregulation of Collagen type 1 a (COL1A1) and hyaluronan synthase 3 mRNA expression and the decrease of Matrix metalloproteinase 1 (MMP-1) and Interleukin-1 beta (IL-1ß) mRNA expression reflected its wrinkle improvement, moisturizing and anti-inflammation function. CONCLUSION: "CELLADEPP Patch", the MTS combined with the iontophoresis technique, exhibits its effectiveness in moisturizing, skin elasticity improvement, and anti-inflammatory function when applied to ex vivo human-derived skin tissue models in experiments. The study has contributed to the understanding of the "CELLADEPP Patch" and laid the foundation for subsequent animal experiments and clinical trials.

Assessment of inflammatory suppression and fibroblast infiltration in tissue remodelling by supercritical CO2 acellular dermal matrix (scADM) utilizing Sprague Dawley models.

Human skin-derived ECM aids cell functions but can trigger immune reactions; therefore it is addressed through decellularization. Acellular dermal matrices (ADMs), known for their regenerative properties, are used in tissue and organ regeneration. ADMs now play a key role in plastic and reconstructive surgery, enhancing aesthetics and reducing capsular contracture risk. Innovative decellularization with supercritical carbon dioxide preserves ECM quality for clinical use. The study investigated the cytotoxicity, biocompatibility, and anti-inflammatory properties of supercritical CO2 acellular dermal matrix (scADM) in vivo based on Sprague Dawley rat models. Initial experiments in vitro with fibroblast cells confirmed the non-toxic nature of scADM and demonstrated cell infiltration into scADMs after incubation. Subsequent tests in vitro revealed the ability of scADM to suppress inflammation induced by lipopolysaccharides (LPS) presenting by the reduction of pro-inflammatory cytokines TNF-α, IL-6, IL-1ß, and MCP-1. In the in vivo model, histological assessment of implanted scADMs in 6 months revealed a decrease in inflammatory cells, confirmed further by the biomarkers of inflammation in immunofluorescence staining. Besides, an increase in fibroblast infiltration and collagen formation was observed in histological staining, which was supported by various biomarkers of fibroblasts. Moreover, the study demonstrated vascularization and macrophage polarization, depicting increased endothelial cell formation. Alteration of matrix metalloproteinases (MMPs) was analyzed by RT-PCR, indicating the reduction of MMP2, MMP3, and MMP9 levels over time. Simultaneously, an increase in collagen deposition of collagen I and collagen III was observed, verified in immunofluorescent staining, RT-PCR, and western blotting. Overall, the findings suggested that scADMs offer significant benefits in improving outcomes in implant-based procedures as well as soft tissue substitution.

Effects of human recombinant granulocyte-colony stimulating factor treatment during in vitro culture on porcine pre-implantation embryos.

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.

Imperatorin Ameliorates the Aging-Associated Porcine Oocyte Meiotic Spindle Defects by Reducing Oxidative Stress and Protecting Mitochondrial Function.

Imperatorin (IMP) exhibits a variety of pharmacological properties, including antioxidant, anti-inflammatory, antibacterial, anti-cancer, and anti-hypertension activities. However, its effects on animal reproduction systems, especially oocyte development, maturation, and aging are not yet clear. In this study, the effects of IMP on oocyte development and aging as well as the underlying molecular mechanisms were explored. Oocytes were cultured for an additional 24 h for aging. Results revealed that the blastocyst formation and hatching rates of embryos, which were parthenogenetically activated aged oocytes, were significantly increased with IMP treatment (40 µM). Simultaneously, well-distributed cortical granules but no significant difference in zona pellucida hardness were observed after IMP treatment. During this stage, intracellular reactive oxygen species, apoptosis, and autophagy levels were decreased, while mitochondrial membrane potential, glutathione level, and activity of superoxide dismutase and catalase were increased. IMP-treated aged oocytes also showed significantly higher expression of MOS, CCNB1, BMP15, and GDF9 than non-IMP-treated aged oocytes although their levels were still lower than those in the fresh oocytes. These results suggest that IMP can effectively ameliorate the quality of aged porcine oocytes by reducing oxidative stress and protecting mitochondrial function.

A Maternal Transcription Factor, Junction Mediating and Regulatory Protein is Required for Preimplantation Development in the Mouse.

Junction-mediating and regulatory protein (JMY) is a regulator of both transcription and actin filament assembly. The actin-regulatory activity of JMY is based on a cluster of three actin-binding Wiskott-Aldrich syndrome protein homology 2 (WH2) domains that nucleate actin filaments directly and promote nucleation of the Arp2/3 complex. In addition to these activities, we examined the activity of JMY generation in early embryo of mice carrying mutations in the JMY gene by CRISPR/Cas9 mediated genome engineering. We demonstrated that JMY protein shuttled expression between the cytoplasm and the nucleus. Knockout of exon 2, CA (central domain and Arp2/3-binding acidic domain) and NLS-2 (nuclear localization signal domain) on the JMY gene by CRISPR/Cas9 system was effective and markedly impeded embryonicdevelopment. Additionally, it impaired transcription and zygotic genome activation (ZGA)-related genes. These results suggest that JMY acts as a transcription factor, which is essential for the early embryonic development in mice.

Autophagy is required for proper meiosis of porcine oocytes maturing in vitro.

Autophagy is an essential cellular mechanism that degrades cytoplasmic proteins and organelles to recycle their components; however, the contribution of autophagy during meiosis has not been studied in porcine oocytes maturing in vitro. In this study, we observed that the autophagy-related gene, LC3, was expressed in porcine oocytes during maturation for 44 h in vitro. Knockdown of the autophagy-related gene, BECN1, reduced both BECN1 and LC3 protein expression levels. Moreover, BECN1 knockdown and treatment with the autophagy inhibitor, LY294002, during maturation of porcine oocytes in vitro impaired polar body extrusion, disturbed mitochondrial function, triggered the DNA damage response, and induced early apoptosis in porcine oocytes. Autophagy inhibition during oocyte maturation also impaired the further developmental potential of porcine oocytes. These results indicate that autophagy is required for the in vitro maturation of porcine oocytes.

Laminarin improves developmental competence of porcine early stage embryos by inhibiting oxidative stress.

Laminarin (LMA), a ß-glucan mixture with good biocompatibility, improves the growth performance and immune response when used as food additives and nutraceuticals. The aim of the present research was to explore the effects of LMA on porcine early stage embryo development, as well as the underlying mechanisms. The results showed that the developmental competence of porcine early stage embryos was dramatically improved after LMA supplementation during the in vitro culture period. The presence of 20 µg/mL LMA during the in vitro culture period significantly improved cleavage rate, blastocyst formation rates, hatching rate, and total cell number in the blastocyst compared to that in the control group. Notably, LMA attenuated the intracellular reactive oxygen species generation induced by H2O2. Furthermore, LMA not only increased intracellular glutathione levels, but also ameliorated mitochondrial membrane potential. In addition, the expression of a zygotic genome activation related gene (YAP1), pluripotency-related genes (OCT4, NANOG, and SOX2), and hatching-related genes (COX2, GATA4, and ITGA5) were up-regulated following LMA supplementation during porcine early stage embryo development. These results demonstrate that LMA has beneficial effects on the development of porcine early stage embryos via regulation of oxidative stress. This evidence provides a novel method for embryo development improvement associated with exposure to LMA.

C-Phycocyanin supplementation during in vitro maturation enhances pre-implantation developmental competence of parthenogenetic and cloned embryos in pigs.

C-Phycocyanin (C-PC), a protein from green microalgae, has been suggested to possess various biological activities, including antioxidant and free radical scavenging properties. The aim of the current study was to explore the effects of C-PC on the maturation of porcine oocytes and subsequent developmental competence after parthenogenetic activation and somatic cell nuclear transfer (SCNT) as well as the underlying mechanisms. There was no significant improvement in nuclear maturation rates between the control and C-PC supplementation groups (1, 3, 5, 10 µg/mL). However, supplementation of 5 µg/mL C-PC in the maturation medium significantly increased blastocyst formation and hatching rates after parthenogenetic activation (59.6 ± 3.6% and 33.0 ± 2.6% vs. 49.8 ± 3.5% and 27.4 ± 2.4%, respectively). In addition, the presence of C-PC during the maturation period significantly improved blastocyst formation rates and total cell numbers after SCNT (24.8 ± 1.9% and 42.2 ± 3.3 vs. 21.6 ± 2.2% and 39.5 ± 3.4, respectively) compared to the control group. Furthermore, cellular proliferation and the expression of pluripotency-related genes (SOX2 and NANOG) were increased in cloned blastocysts derived from the C-PC supplemented group. Importantly, C-PC supplementation during maturation not only improved cumulus expansion and increased the expression of cumulus expansion-related genes (HAS2, PTX3, and PTGS2), but also enhanced antioxidant capacity, improved mitochondria function, and decreased cathepsin B activity in porcine oocytes. These results demonstrate that C-PC may be useful for improving porcine oocyte quality and subsequent developmental competence in embryos.

Melatonin enhances the developmental competence of porcine somatic cell nuclear transfer embryos by preventing DNA damage induced by oxidative stress.

Melatonin has antioxidant and scavenger effects in the cellular antioxidant system. This research investigated the protective effects and underlying mechanisms of melatonin action in porcine somatic cell nuclear transfer (SCNT) embryos. The results suggested that the developmental competence of porcine SCNT embryos was considerably enhanced after melatonin treatment. In addition, melatonin attenuated the increase in reactive oxygen species levels induced by oxidative stress, the decrease in glutathione levels, and the mitochondrial dysfunction. Importantly, melatonin inhibited phospho-histone H2A.X (γH2A.X) expression and comet tail formation, suggesting that γH2A.X prevents oxidative stress-induced DNA damage. The expression of genes involved in homologous recombination and non-homologous end-joining pathways for the repair of double-stranded breaks (DSB) was reduced upon melatonin treatment in porcine SCNT embryos at day 5 of development under oxidative stress condition. These results indicated that melatonin promoted porcine SCNT embryo development by preventing oxidative stress-induced DNA damage via quenching of free radical formation. Our results revealed a previously unrecognized regulatory effect of melatonin in response to oxidative stress and DNA damage. This evidence provides a novel mechanism for the improvement in SCNT embryo development associated with exposure to melatonin.

Toxic effects of atrazine on porcine oocytes and possible mechanisms of action.

Because atrazine is a widely used herbicide, its adverse effects on the reproductive system have been extensively researched. In this study, we investigated the effects of atrazine exposure on porcine oocyte maturation and the possible mechanisms. Our results showed that the rates of oocyte maturation significantly decreased after treatment with 200 µM atrazine in vitro. Atrazine treatment resulted in abnormal spindle morphology but did not affect actin distribution. Atrazine exposure not only triggered a DNA damage response but also decreased MPF levels in porcine oocytes. Our results also revealed that atrazine worsened porcine oocyte quality by causing excessive accumulation of superoxide radicals, increasing cathepsin B activity, and decreasing the GSH level and mitochondrial membrane potential. Furthermore, atrazine decreased developmental competence of porcine oocytes up to the blastocyst stage and changed some properties: cell numbers, apoptosis, and related gene expression levels. Collectively, our results indicate that porcine oocyte maturation is defective after atrazine treatment at least through disruption of spindle morphology, MPF activity, and mitochondrial function and via induction of DNA damage, which probably reduces developmental competence.

Effects of antifreeze glycoprotein 8 (AFGP8) supplementation during vitrification on the in vitro developmental capacity of expanded bovine blastocysts.

Cryopreservation is an effective method for the long-term storage of valuable germplasm in the field of reproductive research. The present study examined the developmental capacity of post-thaw bovine blastocysts during vitrification after supplementation with antifreeze glycoprotein 8 (AFGP8). Survival and re-expansion rates in culture during the 12h after thawing were significantly higher in the AFGP8-treated than untreated group. In addition, blastocysts from the AFGP8-treated group exhibited lower rates of apoptosis. Real-time reverse transcription-polymerase chain reaction analysis showed that the expression of the Bcl-2 gene, coding for an anti-apoptotic protein, was increased significantly, whereas the expression of the pro-apoptotic gene Bax was decreased significantly in the AFGP8-treated group. The cellular proliferation rate and mitochondrial membrane potential were significantly higher in post-thaw re-expanded blastocysts from the AFGP8-treated compared with untreated group. In addition, outgrowth potential in post-thaw blastocysts in re-expanded blastocysts after vitrification was significantly increased in the AFGP8-treated compared with untreated group. Together, these results are the first to demonstrate that the addition of AFGP8 during vitrification can help protect bovine blastocysts against chill-induced injury.

The Role of Glucose Metabolism on Porcine Oocyte Cytoplasmic Maturation and Its Possible Mechanisms.

In the present study, we investigated the potential role of glucose and pyruvate in the cytoplasmic maturation of porcine oocytes by investigating the effect of glucose and/or pyruvate supplementation, in the presence or absence of 10% porcine follicular fluid (PFF), on meiotic maturation and subsequent embryo development. In the absence of 10% PFF, without exogenous addition of glucose and pyruvate, the medium seemed unable to support maturation. In the presence of 10% PFF, the addition of 5.6 mM glucose and/or 2 mM pyruvate during in vitro maturation of cumulus enclosed oocytes increased MII oocyte and blastocyst rates. In contrast, oocytes denuded of cumulus cells were not able to take full advantage of the glucose in the medium, as only pyruvate was able to increase the MII rate and the subsequent early embryo developmental ability. Treatment of cumulus enclosed oocytes undergoing maturation with 200 µM dehydroepiandrosterone (DHEA), a pentose phosphate pathway inhibitor, or 2 µM iodoacetate (IA), a glycolysis inhibitor, significantly reduced GHS, intra-oocyte ATP, maternal gene expression, and MPF activity levels. DHEA was also able to increase ROS and reduce the levels of NADPH. Moreover, blastocysts of the DHEA- or IA-treated groups presented higher apoptosis rates and markedly lower cell proliferation cell rates than those of the non-treated group. In conclusion, our results suggest that oocytes maturing in the presence of 10% PFF can make full use of energy sources through glucose metabolism only when they are accompanied by cumulus cells, and that pentose phosphate pathway (PPP) and glycolysis promote porcine oocyte cytoplasmic maturation by supplying energy, regulating maternal gene expression, and controlling MPF activity.

Progesterone influences cytoplasmic maturation in porcine oocytes developing in vitro.

Progesterone (P4), an ovarian steroid hormone, is an important regulator of female reproduction. In this study, we explored the influence of progesterone on porcine oocyte nuclear maturation and cytoplasmic maturation and development in vitro. We found that the presence of P4 during oocyte maturation did not inhibit polar body extrusions but significantly increased glutathione and decreased reactive oxygen species (ROS) levels relative to that in control groups. The incidence of parthenogenetically activated oocytes that could develop to the blastocyst stage was higher (p < 0.05) when oocytes were exposed to P4 as compared to that in the controls. Cell numbers were increased in the P4-treated groups. Further, the P4-specific inhibitor mifepristone (RU486) prevented porcine oocyte maturation, as represented by the reduced incidence (p < 0.05) of oocyte first polar body extrusions. RU486 affected maturation promoting factor (MPF) activity and maternal mRNA polyadenylation status. In general, these data show that P4 influences the cytoplasmic maturation of porcine oocytes, at least partially, by decreasing their polyadenylation, thereby altering maternal gene expression.

An Improved System for Generation of Diploid Cloned Porcine Embryos Using Induced Pluripotent Stem Cells Synchronized to Metaphase.

Pigs provide outstanding models of human genetic diseases due to their striking similarities with human anatomy, physiology and genetics. Although transgenic pigs have been produced using genetically modified somatic cells and nuclear transfer (SCNT), the cloning efficiency was extremely low. Here, we report an improved method to produce diploid cloned embryos from porcine induced pluripotent stem cells (piPSCs), which were synchronized to the G2/M stage using a double blocking method with aphidicolin and nocodazole. The efficiency of this synchronization method on our piPSC lines was first tested. Then, we modified our traditional SCNT protocol to find a workable protocol. In particular, the removal of a 6DMAP treatment post-activation enhanced the extrusion rate of pseudo-second-polar bodies (p2PB) (81.3% vs. 15.8%, based on peak time, 4hpa). Moreover, an immediate activation method yielded significantly more blastocysts than delayed activation (31.3% vs. 16.0%, based on fused embryos). The immunofluorescent results confirmed the effect of the 6DMAP treatment removal, showing remarkable p2PB extrusion during a series of nuclear transfer procedures. The reconstructed embryos from metaphase piPSCs with our modified protocol demonstrated normal morphology at 2-cell, 4-cell and blastocyst stages and a high rate of normal karyotype. This study demonstrated a new and efficient way to produce viable cloned embryos from piPSCs when synchronized to the G2/M phase of the cell cycle, which may lead to opportunities to produce cloned pigs from piPSCs more efficiently.

Autophagy and ubiquitin-mediated proteolysis may not be involved in the degradation of spermatozoon mitochondria in mouse and porcine early embryos.

The mitochondrial genome is maternally inherited in animals, despite the fact that paternal mitochondria enter oocytes during fertilization. Autophagy and ubiquitin-mediated degradation are responsible for the elimination of paternal mitochondria in Caenorhabditis elegans; however, the involvement of these two processes in the degradation of paternal mitochondria in mammals is not well understood. We investigated the localization patterns of light chain 3 (LC3) and ubiquitin in mouse and porcine embryos during preimplantation development. We found that LC3 and ubiquitin localized to the spermatozoon midpiece at 3 h post-fertilization, and that both proteins were colocalized with paternal mitochondria and removed upon fertilization during the 4-cell stage in mouse and the zygote stage in porcine embryos. Sporadic paternal mitochondria were present beyond the morula stage in the mouse, and paternal mitochondria were restricted to one blastomere of 4-cell embryos. An autophagy inhibitor, 3-methyladenine (3-MA), did not affect the distribution of paternal mitochondria compared with the positive control, while an autophagy inducer, rapamycin, accelerated the removal of paternal mitochondria compared with the control. After the intracytoplasmic injection of intact spermatozoon into mouse oocytes, LC3 and ubiquitin localized to the spermatozoon midpiece, but remnants of undegraded paternal mitochondria were retained until the blastocyst stage. Our results show that paternal mitochondria colocalize with autophagy receptors and ubiquitin and are removed after in vitro fertilization, but some remnants of sperm mitochondrial sheath may persist up to morula stage after intracytoplasmic spermatozoon injection (ICSI).

Artificial control maturation of porcine oocyte by dibutyryl cyclicAMP.

In this study, we investigated the effects of various durations of dibutyryl cyclic AMP (dbcAMP) treatment on the in vitro maturation (IVM) and subsequent development of parthenogenetically activated embryos. Immature porcine oocytes were cultured with or without 1 mM dbcAMP during the first 20, 28, or 36 h of culture, and then incubated for an additional 24 h without dbcAMP. The expression of Wee1B, Myt, and Cdc25B and the level of maturation promoting factor (MPF) in metaphase II oocytes were analyzed by real-time PCR (qRT-PCR) and enzyme linked immunosorbent assay (ELISA), respectively. The distribution of actin microfilaments in oocytes was also assessed. Subsequently, apoptotic cells in blastocysts from each group were visualized by transferase-mediated dUTP nick-end labeling staining. Results showed that oocytes extruded the first polar body between 12 and 18 h after being released from dbcAMP. MPF activity in oocytes at 28 + 24 h and 36 + 24 h after dbcAMP treatment was higher than that in the control group. Significantly more blastocysts were present among embryos in 28 + 24 h (54.28% vs. 39.11%, P < 0.05) and 36 + 24 h (47.24% vs. 32.94%, P < 0.05) groups than among embryos cultured in the absence of dbcAMP. However, the number of total and apoptotic cells was not significantly different between groups. The distribution of actin microfilaments was abnormal in oocytes cultured for 60 h without dbcAMP. In addition, the expression of Wee1B, Myt, and Cdc25B was higher in the control group at 44 h than in the dbcAMP group, but there were no differences in expression at the other time points. In conclusion, dbcAMP treatment delays oocyte maturation and maintains oocyte quality.

The association of mitochondrial potential and copy number with pig oocyte maturation and developmental potential.

ATP is critical for oocyte maturation, fertilization, and subsequent embryo development. Both mitochondrial membrane potential and copy number expand during oocyte maturation. In order to differentiate the roles of mitochondrial metabolic activity and mtDNA copy number during oocyte maturation, we used two inhibitors, FCCP (carbonyl cyanide p-(tri-fluromethoxy)phenyl-hydrazone) and ddC (2'3-dideoxycytidine), to deplete the mitochondrial membrane potential (Δφm) and mitochondrial copy number, respectively. FCCP (2000 nM) reduced ATP production by affecting mitochondrial Δφm, decreased the mRNA expression of Bmp15 (bone morphogenetic protein 15), and shortened the poly(A) tails of Bmp15, Gdf9 (growth differentiation factor 9), and Cyclin B1 transcripts. FCCP (200 and 2000 nM) also affected p34(cdc2) kinase activity. By contrast, ddC did not alter ATP production. Instead, ddC significantly decreased mtDNA copy number (P < 0.05). FCCP (200 and 2000 nM) also decreased extrusion of the first polar body, whereas ddC at all concentrations did not affect the ability of immature oocytes to reach metaphase II. Both FCCP (200 and 2000 nM) and ddC (200 and 2000 µM) reduced parthenogenetic blastocyst formation compared with untreated oocytes. However, these inhibitors did not affect total cell number and apoptosis. These findings suggest that mitochondrial metabolic activity is critical for oocyte maturation and that both mitochondrial metabolic activity and replication contribute to the developmental competence of porcine oocytes.

Production of pigs expressing a transgene under the control of a tetracycline-inducible system.

Pigs are anatomically and physiologically closer to humans than other laboratory animals. Transgenic (TG) pigs are widely used as models of human diseases. The aim of this study was to produce pigs expressing a tetracycline (Tet)-inducible transgene. The Tet-on system was first tested in infected donor cells. Porcine fetal fibroblasts were infected with a universal doxycycline-inducible vector containing the target gene enhanced green fluorescent protein (eGFP). At 1 day after treatment with 1 µg/ml doxycycline, the fluorescence intensity of these cells was increased. Somatic cell nuclear transfer (SCNT) was then performed using these donor cells. The Tet-on system was then tested in the generated porcine SCNT-TG embryos. Of 4,951 porcine SCNT-TG embryos generated, 850 were cultured in the presence of 1 µg/ml doxycycline in vitro. All of these embryos expressed eGFP and 15 embryos developed to blastocyst stage. The remaining 4,101 embryos were transferred to thirty three surrogate pigs from which thirty eight cloned TG piglets were obtained. PCR analysis showed that the transgene was inserted into the genome of each of these piglets. Two TG fibroblast cell lines were established from these TG piglets, and these cells were used as donor cells for re-cloning. The re-cloned SCNT embryos expressed the eGFP transgene under the control of doxycycline. These data show that the expression of transgenes in cloned TG pigs can be regulated by the Tet-on/off systems.

Histone deacetylase inhibitor trichostatin A affects porcine oocyte maturation in vitro.

Previous studies show that porcine oocyte aging resulting from asynchronised IVM impairs embryo developmental competence. In the present study we investigated whether trichostatin A (TSA; an inhibitor of histone deacetylation) prolongs the maturation time and prevents the aging of oocytes. Porcine oocytes were cultured in medium containing increasing concentrations of TSA (300 nM) for 24, 44 or 64 h. The percentage of oocytes that underwent germinal vesicle breakdown was significantly lower in the TSA-treated group (300 nM) than in the control group. TSA did not affect oocyte quality at MII based on levels of maturation-promoting factor, the phosphorylation status of mitogen-activated protein kinase or histone H3K9 acetylation analysis. We also compared the preimplantation developmental competence and the viability of pathenogenetic embryos treated with 100 nM TSA for 24h and then continuously cultured for another 24h in TSA free condition. No significant differences were observed for either parameter between the TSA-treated and control groups. These results indicate that TSA prolongs the IVM of porcine oocytes but that oocyte quality and aging are not affected. These findings provide a feasible option by which to adjust the initiation time of downstream experiments based on porcine matured oocytes.