Optimizing Embryo Collection for Application of CRISPR/Cas9 System and Generation of Fukutin Knockout Rat Using This Method.

Rat animal models are widely used owing to their relatively superior cognitive abilities and higher similarity compared with mouse models to human physiological characteristics. However, their use is limited because of difficulties in establishing embryonic stem cells and performing genetic modifications, and insufficient embryological research. In this study, we established optimal superovulation and fertilized-egg transfer conditions, including optimal hormone injection concentration (≥150 IU/kg of PMSG and hCG) and culture medium (mR1ECM), to obtain high-quality zygotes and establish in vitro fertilization conditions for rats. Next, sgRNA with optimal targeting activity was selected by performing PCR analysis and the T7E1 assay, and the CRISPR/Cas9 system was used to construct a rat model for muscular dystrophy by inducing a deficiency in the fukutin gene without any off-target effect detected. The production of fukutin knockout rats was phenotypically confirmed by observing a drop-in body weight to one-third of that of the control group. In summary, we succeeded in constructing the first muscular dystrophy disease rat model using the CRISPR/CAS9 system for increasing future prospects of producing various animal disease models and encouraging disease research using rats.

Fasudil alleviates the vascular endothelial dysfunction and several phenotypes of Fabry disease.

Fabry disease (FD), a lysosomal storage disorder, is caused by defective α-galactosidase (GLA) activity, which results in the accumulation of globotriaosylceramide (Gb3) in endothelial cells and leads to life-threatening complications such as left ventricular hypertrophy (LVH), renal failure, and stroke. Enzyme replacement therapy (ERT) results in Gb3 clearance; however, because of a short half-life in the body and the high immunogenicity of FD patients, ERT has a limited therapeutic effect, particularly in patients with late-onset disease or progressive complications. Because vascular endothelial cells (VECs) derived from FD-induced pluripotent stem cells display increased thrombospondin-1 (TSP1) expression and enhanced SMAD2 signaling, we screened for chemical compounds that could downregulate TSP1 and SMAD2 signaling. Fasudil reduced the levels of p-SMAD2 and TSP1 in FD-VECs and increased the expression of angiogenic factors. Furthermore, fasudil downregulated the endothelial-to-mesenchymal transition (EndMT) and mitochondrial function of FD-VECs. Oral administration of fasudil to FD mice alleviated several FD phenotypes, including LVH, renal fibrosis, anhidrosis, and heat insensitivity. Our findings demonstrate that fasudil is a novel candidate for FD therapy.

Melatonin improves the meiotic maturation of porcine oocytes by reducing endoplasmic reticulum stress during in vitro maturation.

Under endoplasmic reticulum (ER)-stress conditions, the unfolded protein response (UPR) generates a defense mechanism in mammalian cells. The regulation of UPR signaling is important in oocyte maturation, embryo development, and female reproduction of pigs. Recent studies have shown that melatonin plays an important role as an antioxidant to improve pig oocyte maturation. However, there is no report on the role of melatonin in the regulation of UPR signaling and ER-stress during in vitro maturation (IVM) of porcine oocytes. Therefore, the objective of this study was to investigate the antioxidative effects of melatonin on porcine oocyte maturation through the regulation of ER-stress and UPR signaling. We investigated the changes in the mRNA/protein expression levels of three UPR signal genes (Bip/Grp78, ATF4, P90/50ATF6, sXbp1, and CHOP) on oocytes, cumulus cells, and cumulus-oocyte complexes (COCs) during IVM (metaphase I; 22 hours and metaphase II; 44 hours) by Western blot and reverse transcription-polymerase chain reaction analysis. Treatment with the ER-stress inducer, tunicamycin (Tm), significantly increased expression of UPR markers. Additionally, cumulus cell expansion and meiotic maturation of oocytes were reduced in COCs of Tm-treated groups (1, 5, and 10 µg/mL). We confirmed the reducing effects of melatonin (0.1 µmol/L) on ER-stress after pretreatment with Tm (5 µg/mL; 22 hours) in maturing COCs. Addition of melatonin (0.1 µmol/L) to Tm-pretreated COCs recovered meiotic maturation rates and expression of most UPR markers. In conclusion, we confirmed a role for melatonin in the modulation of UPR signal pathways and reducing ER-stress during IVM of porcine oocytes.

Ganglioside GM3 induces cumulus cell apoptosis through inhibition of epidermal growth factor receptor-mediated PI3K/AKT signaling pathways during in vitro maturation of pig oocytes.

Gangliosides are components of the mammalian plasma membrane that help regulate receptor signaling. Ganglioside GM3, for example, plays an important role in initiating apoptosis in cancer cells; however, physiological roles for GM3 in normal processes, such as during pig oocyte maturation, are not clear. The aim of this study was to investigate the functional link between GM3 and cellular apoptosis in porcine cumulus-oocyte-complexes (COCs) during in vitro maturation. Our results indicated that denuded oocytes possess less ST3GAL5, a GM3-synthesizing enzyme, than cumulus cells or COCs after 44 hr of in vitro maturation. GM3 also affected the meiotic maturation of cultured pig oocytes, as evaluated by orcein staining. In vitro treatment of COCs with exogenous GM3 also reduced cumulus cell expansion, the proportion of meiotic maturation, and increased cumulus cell transcription of PTX3, TNFAIP6, and HAS2. Interestingly, GM3 treatment reduced the expression of Epidermal growth factor receptor (EGFR)-mediated Phosphoinositide 3-kinase/AKT signaling proteins in COCs in a concentration-dependent manner, instead increasing the abundance of pro-apoptotic factors such as AIF, activated Caspase 9, cleaved PARP1, and Caspase 3 were. Thus, GM3 might affect porcine oocyte maturation via suppression of EGFR-mediated PI3K/AKT signaling and/or induction of apoptosis during in vitro maturation.

Knockout of miR-221 and miR-222 reveals common and specific targets for paralogous miRNAs.

MicroRNAs (miRNAs) regulate the expression of mRNA through sequence-specific binding of the 3' untranslated region (UTR). The seed sequence of miRNAs is the key determinant for target site recognition. Paralogous miRNAs, which share the same seed sequences but differ in their 3' regions, are known to regulate largely overlapping groups of mRNAs. However, no study has analyzed functional differences between paralogous miRNAs with proper experimental methods. In this study, we compared the targets of paralogous miRNAs, miR-221 and miR-222. Using a nuclease-mediated genome engineering technique, we established knockout cell lines for these miRNAs, and precisely analyzed differences in target regulation. We found that miR-221 and miR-222 suppress the previously identified targets, CDKN1B and CDKN1C, differentially. Whereas both miRNAs suppressed CDKN1B, only miR-221 suppressed CDKN1C. From transcriptome analyses, we found that several different target mRNAs were regulated by each of miR-221 and miR-222 independently, although a large number of mRNAs responded commonly to miR-221 and miR-222. This is the first study to compare the mRNA regulations by paralogous miRNAs and illustrate that paralogous miRNAs with the same seed sequence also have difference in target regulation.

Requirement of the transcription factor USF1 in bovine oocyte and early embryonic development.

Upstream stimulating factor 1 (USF1) is a basic helix-loop-helix transcription factor that specifically binds to E-box DNA motifs, known cis-elements of key oocyte expressed genes essential for oocyte and early embryonic development. However, the functional and regulatory role of USF1 in bovine oocyte and embryo development is not understood. In this study, we demonstrated that USF1 mRNA is maternal in origin and expressed in a stage specific manner during the course of oocyte maturation and preimplantation embryonic development. Immunocytochemical analysis showed detectable USF1 protein during oocyte maturation and early embryonic development with increased abundance at 8-16-cell stage of embryo development, suggesting a potential role in embryonic genome activation. Knockdown of USF1 in germinal vesicle stage oocytes did not affect meiotic maturation or cumulus expansion, but caused significant changes in mRNA abundance for genes associated with oocyte developmental competence. Furthermore, siRNA-mediated depletion of USF1 in presumptive zygote stage embryos demonstrated that USF1 is required for early embryonic development to the blastocyst stage. A similar (USF2) yet unique (TWIST2) expression pattern during oocyte and early embryonic development for related E-box binding transcription factors known to cooperatively bind USF1 implies a potential link to USF1 action. This study demonstrates that USF1 is a maternally derived transcription factor required for bovine early embryonic development, which also functions in regulation of JY1, GDF9, and FST genes associated with oocyte competence.

Functional role of the bovine oocyte-specific protein JY-1 in meiotic maturation, cumulus expansion, and subsequent embryonic development.

Oocyte-expressed genes regulate key aspects of ovarian follicular development and early embryogenesis. We previously demonstrated a requirement of the oocyte-specific protein JY-1 for bovine early embryogenesis. Given that JY-1 is present in oocytes throughout folliculogenesis, and oocyte-derived JY-1 mRNA is temporally regulated postfertilization, we hypothesized that JY-1 levels in oocytes impact nuclear maturation and subsequent early embryogenesis. A novel model system, whereby JY-1 small interfering RNA was microinjected into cumulus-enclosed germinal vesicle-stage oocytes and meiotic arrest maintained for 48 h prior to in vitro maturation (IVM), was validated and used to determine the effect of reduced oocyte JY-1 expression on nuclear maturation, cumulus expansion, and embryonic development after in vitro fertilization. Depletion of JY-1 protein during IVM effectively reduced cumulus expansion, percentage of oocytes progressing to metaphase II, proportion of embryos that cleaved early, total cleavage rates and development to 8- to 16-cell stage, and totally blocked development to the blastocyst stage relative to controls. Supplementation with JY-1 protein during oocyte culture rescued effects of JY-1 depletion on meiotic maturation, cumulus expansion, and early cleavage, but did not rescue development to 8- to 16-cell and blastocyst stages. However, effects of JY-1 depletion postfertilization on development to 8- to 16-cell and blastocyst stages were rescued by JY-1 supplementation during embryo culture. In conclusion, these results support an important functional role for oocyte-derived JY-1 protein during meiotic maturation in promoting progression to metaphase II, cumulus expansion, and subsequent embryonic development.

Industrialization possibilities of purified pig sperm hyaluronidase.

The goals of the present study were to develop a simple method for obtain highly purified pig sperm hyaluronidase (pHyase) and to assess its activity, function, and safety. In mammals, sperm-specific glycophosphatidylinositol (GPI)-anchored Hyase assists sperm penetration through the cumulus mass surrounding the egg and aids in the dispersal of the cumulus-oocyte complex. Recently, Purified bovine sperm hyaluronidase (bHyase) has been shown to enhance therapeutic drug transport by breaking down the hyaluronan barrier to the lymphatic and capillary vessels, thereby facilitating tissue absorption. Commercially available Hyase is typically isolated from bovine or ovine; which have several disadvantages, including the risk of bovine spongiform encephalopathy, low homology with human Hyase, and the requirement for relatively complex isolation procedures. This study successfully isolated highly purified pHyase in only two steps, using ammonium sulfate precipitation and fast protein liquid chromatography. The isolated Hyase had activity equal to that of commercial bHyase, facilitated in vitro fertilization, and effectively dissolved high molecule hyaluronic acid. This simple, effective isolation method could improve the availability of pHyase for research and clinical applications.

Regulation of angiogenesis-related prostaglandin f2alpha-induced genes in the bovine corpus luteum.

We recently compared prostaglandin F2alpha (PG)-induced global gene expression profiles in PG-refractory, bovine corpus luteum (CL) collected on Day 4 of the estrous cycle, versus PG-responsive, Day 11 CL. Transcriptome analyses led us to study the regulation of angiogenesis-related genes by PG and their functions in luteal endothelial cells (ECs). We found that PG regulated angiogenesis-modulating factors in a luteal stage-dependent way. A robust increase in FGF2 expression (mRNA and protein) occurred in the PG-refractory Day 4 CL promoting CL survival and function. Inhibitors of FGF2 action, thrombospondin 1 and 2, their receptor (CD36), and PTX3 were upregulated by PG specifically in Day 11 CL undergoing luteolysis. VEGF mRNA decreased 4 h post-PG in both Day 4 and Day 11 CL. The resulting destabilization of blood vessels in Day 11 CL is expected to weaken the gland and reduce its hormonal output. These genes were expressed in dispersed luteal ECs and steroidogenic cells; however, thrombospondin 1 and FGF2 were more abundant in luteal ECs. Expression of such genes and their ability to modulate FGF2 actions were investigated. Similar to its in vivo effect, PG, in vitro, stimulated the expression of thrombospondins and PTX3 genes in several luteal cell models. Importantly, these factors influenced the angiogenic properties of luteal ECs. FGF2 dose-dependently enhanced cell migration and proliferation, whereas thrombospondin 1 and PTX3 inhibited FGF2 actions in luteal ECs. Collectively, the data presented here suggest that, by tilting the balance between pro- and antiangiogenic factors, PG can potentially control the ability of the CL to resist or advance toward luteolysis.

Hyaluronidase 6 Does Not Affect Cumulus-Oocyte Complex Dispersal and Male Mice Fertility.

Glycosylphosphatidylinositol-anchored sperm hyaluronidases (HYAL) assist sperm penetration through the cumulus-oocyte complex (COC), but their role in mammalian fertilization remains unclear. Previously, we demonstrated that sperm from HYAL 5 and 7 double-knockout (dKO) mice produced significantly less offspring than sperm from wild-type mice due to defective COC dispersal. However, the HYAL6 gene remained active in the sperm from the dKO mice, indicating that they were not entirely infertile. This study explored the role of HYAL6 in fertilization by analyzing HYAL6-mutant mice. In this mouse model, HYAL5 and HYAL7 were present in the HYAL6-knockout sperm, and they could disperse hyaluronic acid. We found that HYAL6 was present on the surface of sperm. However, male mice lacking the HYAL6 gene had normal fertility, testicular integrity, and sperm characteristics. Furthermore, in vitro fertilization assays demonstrated that HYAL6-deficient epididymal sperm functioned normally. Therefore, HYAL6 is dispensable for fertilization.

Sperm hyaluronidase is critical to mammals' fertilization for its ability to disperse cumulus-oocyte complex layer.

Glycosylphosphatidylinositol-anchored sperm hyaluronidases have long been believed to assist in sperm penetration through the cumulus-oocyte complex (COC); however, their role in mammalian fertilization remains unclear. Previously, we have shown that hyaluronidase 5 (Hyal5)/Hyal7 double-knockout (dKO) mice produce significantly fewer offspring than their wild-type (WT) counterparts because of defective COC dispersal. Male infertility is mainly caused by a low sperm count. It can be further exacerbated by the deficiency of sperm hyaluronidase, which disperses the cumulus cells of the outer layer of the COC. In the current study, we evaluated the effects of a low count of Hyal-deficient sperm and conditions of ovulated oocytes on the fertilization rate using a mouse model. Our results demonstrated that a low sperm count further decreases the in vitro fertilization (IVF) rate of Hyal-deficient dKO spermatozoa. In addition, the dKO spermatozoa resulted in a fertilization rate of 12.5% upon fertilizing COCs with a thick cumulus layer, whereas the IVF rate was comparable to that of WT spermatozoa when oocytes with a thin or no cumulus layer were fertilized. Finally, we proved that the IVF rate of dKO spermatozoa could be recovered by adding rat spermatozoa as a source of sperm hyal. Our results suggest that a deficiency of proteins involved in fertilization, such as sperm hyal, has a vital role in fertilization.

MicroRNA-196a regulates bovine newborn ovary homeobox gene (NOBOX) expression during early embryogenesis.

BACKGROUND: Oocyte-derived maternal RNAs drive early embryogenesis when the newly formed embryo is transcriptionally inactive. Recent studies in zebrafish have identified the role of microRNAs during the maternal-to-embryonic transition (MET). MicroRNAs are short RNAs that bind to the 3' UTR of target mRNAs to repress their translation and accelerate their decay. Newborn ovary homeobox gene (NOBOX) is a transcription factor that is preferentially expressed in oocytes and essential for folliculogenesis in mice. NOBOX knockout mice are infertile and lack of NOBOX disrupts expression of many germ-cell specific genes and microRNAs. We recently reported the cloning and expression of bovine NOBOX during early embryonic development and our gene knockdown studies indicate that NOBOX is a maternal effect gene essential for early embryonic development. As NOBOX is a maternal transcript critical for development and NOBOX is depleted during early embryogenesis, we hypothesized that NOBOX is targeted by microRNAs for silencing and/or degradation. RESULTS: Using an algorithm "MicroInspector", a potential microRNA recognition element (MRE) for miR-196a was identified in the 3' UTR of the bovine NOBOX mRNA. Expression analysis of miR-196a in bovine oocytes and during early embryonic development indicated that it is expressed both in oocytes and embryos and tends to increase at the four-cell and eight-cell stages. Ectopic expression of NOBOX and miR-196a in HeLa cells inhibited the expression of NOBOX protein compared to the control cells without miR-196a. Similarly, the activity of a luciferase construct containing the entire 3' UTR of bovine NOBOX was suppressed, and the regulation was abolished by mutations in the miR-196a binding site indicating that the predicted MRE is critical for the direct and specific binding of miR-196a to the NOBOX mRNA. Furthermore, ectopic expression of miR-196a mimic in bovine early embryos significantly reduced the NOBOX expression at the both mRNA and protein levels. CONCLUSION: Collectively, our results demonstrate that miR-196a is a bona fide negative regulator of NOBOX during bovine early embryogenesis.

Obtusifolin, an Anthraquinone Extracted from Senna obtusifolia (L.) H.S.Irwin & Barneby, Reduces Inflammation in a Mouse Osteoarthritis Model.

Osteoarthritis (OA) is an age-related degenerative disease that causes cartilage dysfunction and inflammation. Obtusifolin, an anthraquinone extracted from Senna obtusifolia (L.) H.S.Irwin & Barneby seeds, has anti-inflammatory functions; it could be used as a drug component to relieve OA symptoms. In this study, we investigated the effects of obtusifolin on OA inflammation. In vitro, interleukin (IL)-1ß (1 ng/mL)-treated mouse chondrocytes were co-treated with obtusifolin at different concentrations. The expression of matrix metalloproteinase (Mmp) 3, Mmp13, cyclooxygenase 2 (Cox2), and signaling proteins was measured by polymerase chain reaction and Western blotting; collagenase activity and the PGE2 level were also determined. In vivo, OA-induced C57BL/6 mice were administered obtusifolin, and their cartilage was stained with Safranin O to observe damage. Obtusifolin inhibited Mmp3, Mmp13, and Cox2 expression to levels similar to or more than those after treatment with celecoxib. Additionally, obtusifolin decreased collagenase activity and the PGE2 level. Furthermore, obtusifolin regulated OA via the NF-κB signaling pathway. In surgically induced OA mouse models, the cartilage destruction decreased when obtusifolin was administered orally. Taken together, our results show that obtusifolin effectively reduces cartilage damage via the regulation of MMPs and Cox2 expression. Hence, we suggest that obtusifolin could be a component of another OA symptom reliever.

A cell function study on calcium regulation of a novel calcium-sensing receptor mutation (p.Tyr825Phe).

PURPOSE: Autosomal dominant hypocalcemia with hypercalciuria is a genetic disease characterized by hypoparathyroidism with hypercalciuria. We discovered a novel variant (p.Tyr825Phe[Y825F]) of the CASR gene in a neonate with congenital hypoparathyroidism and hypercalciuria and conducted a cell function study to determine whether the CASR-Y825F variant was pathogenic. METHODS: To perform a functional study on CaSR-Y825F, we constructed expression vectors expressing wild-type (WT) CASR and CASR-Y825F. After transfection of each expression vector into HEK293 cells, we examined alterations in intracellular signaling. Mitogen-activated protein kinase (MAPK) signaling activity of HEK293 cells expressing CASR-WT or CASR-Y825F was determined. Changes in intracellular calcium ions ([Ca2+]i) by extracellular calcium ion ([Ca2+]e) stimulation were quantitatively compared and analyzed. RESULTS: Cells expressing CASR-Y825F showed elevated of MAPK signaling (phospho-ERK [pERK], phospho-JNK [pJNK], phospho-p38 [pp38]) and increased [Ca2+]i levels at low [Ca2+]e stimulation compared with cells expressing CASR-WT. Additionally, [Ca2+]i levels in HEK293 cells expression CASR-WT and CASR-Y825F were determined at 340 nm/380 nm wavelength ratios using Fura-2 AM. At [Ca2+]e concentrations of 2.5 mM and 3 mM, the ratios of CASR-Y825F cells were higher (2.6 and 3.5, respectively) than those of CASR-WT cells (1.04 and 1.40, respectively). CONCLUSION: This cell function study proved that the CASR-Y825F expressed in HEK293 cells elevated MAPK signaling (pERK, pJNK, pp38) and increased [Ca2+]i to induce hypocalcemia.

V-Set and Immunoglobulin Domain-Containing 1 (VSIG1), Predominantly Expressed in Testicular Germ Cells, Is Dispensable for Spermatogenesis and Male Fertility in Mice.

To elucidate the functional role of V-set and immunoglobulin domain-containing 1 (VSIG1) in spermatogenesis and fertilization, we knocked out (KO) VSIG1 in a mouse embryo using CRISPR/Cas9 (Clustered regularly interspaced short palindromic repeat/CRISPR-associated protein 9) -mediated genome editing. Reverse transcription PCR was performed using cDNA synthesized from VSIG1 KO testis RNA. Although Western blot analysis using a specific antibody to VSIG1 confirmed VSIG1 protein defects in the KO mice, hematoxylin-eosin staining analysis was similar in the KO and wild-type mice. Additionally, computer-assisted sperm analysis and in vitro fertilization experiments were conducted to confirm the activity and fertilization ability of sperm derived from the KO mouse. Mice lacking VSIG1 were viable and had no serious developmental defects. As they got older, the KO mice showed slightly higher weight loss, male mice lacking VSIG1 had functional testes, including normal sperm number and motility, and both male and female mice lacking VSIG1 were fertile. Our results from VSIG1 KO mice suggest that VSIG1 may not play essential roles in spermatogenesis and normal testis development, function, and maintenance. VSIG1 in sperm is dispensable for spermatogenesis and male fertility in mice. As several genes are known to possess slightly different functions depending on the species, the importance and molecular mechanism of VSIG1 in tissues of other species needs further investigation.

Behaviors of ATP-dependent chromatin remodeling factors during maturation of bovine oocytes in vitro.

The mammalian oocyte undergoes dynamic changes in chromatin structure to reach complete maturation. However, little known is about behaviors of ATP-dependent chromatin remodeling factors (ACRFs) during meiosis. Here, we found that respective ACRFs may differently behave in the process of oocyte maturation in the bovine. All ACRFs interacted with oocytic chromatin at the germinal vesicle (GV) stage. Mi-2 and hSNF2H disappeared from GV-chromatin within 1 hr of in vitro culture whereas Brg-1 and BAF-170 were retained throughout germinal vesicle break down (GVBD). Brg-1 was localized on the condensed chromatin outside, whereas BAF-170 was entirely excluded from condensed chromatin. Thereafter, Brg-1 and BAF-170 interacted with metaphase I and metaphase II chromosomes. These results imply that Mi-2 and hSNF2H may initiate the meiotic resumption, and Brg-1 and BAF-170 may support chromatin condensation during meiosis. In addition, DNA methylation and methylation of histone H3 at lysine 9 (H3K9) seem to be constantly retained in the oocyte chromatin throughout in vitro maturation. Inhibition of ACRF activity by treatment with the inhibitor apyrase led to retarded chromatin remodeling in bovine oocytes, thereby resulting in poor development of fertilized embryos. Therefore, these results indicate that precise behaviors of ACRFs during meiosis are critical for nuclear maturation and subsequent embryonic development in the bovine.

Exogenous Ganglioside GT1b Enhances Porcine Oocyte Maturation, Including the Cumulus Cell Expansion and Activation of EGFR and ERK1/2 Signaling.

Ganglioside GT1b is well-known for its role in cytokine production and in activating epidermal growth factor receptor (EGFR)-mediated signaling pathways in cancer cells. However, there are no reports that clearly elucidate the role of GT1b in EGFR-mediated signaling pathways in porcine oocytes during the process of in vitro maturation (IVM). In this study, we investigated the role of GT1b in EGFR-mediated activation of the ERK1/2 pathway in porcine cumulus-oocyte complexes (COCs) at 44 h of IVM. Our data show that expression of the ST3GAL2 protein significantly increased in porcine COCs at 44 h irrespective of treatment with EGF. Meiotic maturation and mRNA levels of factors (HAS2, TNFAIP6, and PTX3) related to cumulus cell expansion significantly increased in COCs treated with 2 µM GT1b during IVM in the absence of EGF. They also increased in COCs treated with EGF/GT1b as compared to that in the other groups. Interestingly, protein levels of EGFR, phospho-EGFR, ERK1/2, and phospho-ERK1/2 dramatically increased in COCs treated with EGF/GT1b. Moreover, the rate of fertilization and the developmental competence of blastocyst were significantly higher in EGF/GT1b-treated COCs. Taken together, these results suggest that exogenous GT1b improves meiotic maturation and cumulus cell expansion in porcine COCs via activation of EGFR-mediated ERK1/2 signaling.

A microfluidic in vitro cultivation system for mechanical stimulation of bovine embryos.

This work demonstrates a novel microfluidic in vitro cultivation system for embryos that improves their development using a partially constricted channel that mimics peristaltic muscle contraction. Conventional photolithography and a PDMS replica molding process were used to make straight or constricted microchannels. To investigate the effects of constriction geometry on embryonic development, different constriction widths of the channel were designed. Bovine embryos were loaded and incubated by simply placing them on a tilting machine to provide embryo movement via gravity. The fertilized embryos were cultivated on the microfluidic in vitro cultivation system until the blastocyst, hatching, or hatched blastocyst stages. To confirm the quality of blastocysts in the microfluidic channel, double staining was performed and compared with bovine embryos cultivated by the conventional droplet method. The proportion of eight-cell development among total embryos in the constricted channel (56.7+/-13.7%; mean+/-SD) was superior to that in the straight channel (23.9+/-11.0%). This suggests that the effect of constriction is vital for the early development of bovine embryos in assisted-reproduction research.

Mito-TEMPO improves development competence by reducing superoxide in preimplantation porcine embryos.

Mito-TEMPO is a well-known mitochondria-specific superoxide scavenger. However, the effect of Mito-TEMPO on porcine embryo development, to our knowledge, has not been studied yet. In the present study, porcine embryos were classified into two groups (G1 and G2) based on the cytoplasm lipid contents at the zygote stage. The development of blastocysts derived from G2 zygotes was reduced (G2:16.2 ± 7.9% vs G1: 26.5 ± 5.9%; 1.6-fold, p < 0.05) compared to those from G1 zygotes. In G2 embryos, the proportion of TUNEL-positive cells was also higher than that of G1 embryos. Superoxide in G2 embryos was significantly increased compared to that in G1 embryos. Mitochondrial membrane potential and ATP production were lower in G2 embryos than in G1 embryos. Phosphorylation of Drp1 at Ser 616 increased in G1 embryos during the cleavage stages compared to that in the zygote but was not significantly different in G2 embryos. Then, the effects of Mito-TEMPO were investigated in G2 embryos. Blastocyst formation rate (G2: 19.1 ± 5.1% vs G2 + Mito-TEMPO: 28.8 ± 4.0%; 1.5-fold, p < 0.05) and mitochondrial aggregation were recovered after superoxide reduction by Mito-TEMPO treatment. Thus, we showed that Mito-TEMPO improves blastocyst development by superoxide reduction in porcine embryos in vitro.

Benzoic Acid Enhances Embryo Implantation through LIF-Dependent Expression of Integrin αVß3 and αVß5.

Embryo implantation is the crucial step for a successful pregnancy. Diverse factors, including adhesion molecules, growth factors, and cytokines are important for embryo implantation through improving endometrial receptivity. Benzoic acid (BA), a component of various plants, has been shown to have antifungal and antioxidant effects. However, the effect of BA on embryo implantation remains unknown. Here, we showed the contribution of BA for the enhancement of endometrial receptivity through the leukemia inhibitory factor (LIF)-dependent increase of integrin αV, ß3, and ß5 expression. Furthermore, in vivo study using a mifepristone-induced implantation failure model showed that BA definitely improves the numbers of implantation embryos. Taken together, we suggest that BA has a novel function for embryo implantation through the up-regulation of LIF-mediated integrins, and may be a candidate for therapeutic medicine to increase the pregnancy rate.