Assisted Hatching Treatment of Piezo-Mediated Small Hole on Zona Pellucida in Morula Stage Embryos Improves Embryo Implantation and Litter Size in Mice.

For in vitro produced embryos generated from in vitro fertilization (IVF) or intracytoplasmic sperm injection (ICSI) procedure, the intra- and extra-environmental factors during in vitro culture have significant impact on latter embryo development and fetus growth. Assisted hatching (AH), an effective approach to facilitate hatchability for in vitro generated embryos, is an essential step for successful embryo implantation in the uterus. However, regarding the different AH methods reported in clinical practice, it is still unknown whether zona pellucida (ZP) broken is based on AH applied in diverse stages of embryos affect implantation and fetal development. Here, piezo-mediated AH treatments were classified into four categories: (1) drilling one small hole (SH) with a diameter of 10 µm on ZP (SH); (2) drilling one large hole (LH) with a diameter of 40 µm on ZP (LH); (3) made a small area with diameter of 40-µm thinner on ZP [small area thinner (ST)]; (4) made a large area with a diameter of 80-µm thinner [large area thinner (LT)]. These four AH treatments were applied in different stage embryos including two-cell, four-cell, and morula. The most efficient AH approach was chosen according to the final hatch rate at 120 h after fertilization. We found that the approach of SH applied in morula-stage embryos obtained the highest hatch rate. To further investigate if this treatment has any side effect on later development after embryo transfer, we evaluated embryo implantation, gestational period, litter size, and growth. Our results showed that SH applied in morula-stage embryos could facilitate the implantation process and increase litter size. Meanwhile, this approach had no side effect on birth weight, growth, or gender ratio in the offspring. We conclude that drilling a SH on ZP in morula-stage embryos is an effective and reliable AH approach for in vitro cultured embryos in rodent. And this approach is worth further investigating in human-assisted reproductive technology.

Combined use of dbcAMP and IBMX minimizes the damage induced by a long-term artificial meiotic arrest in mouse germinal vesicle oocytes.

Phosphodiesterase (PDE)-mediated reduction of cyclic adenosine monophosphate (cAMP) activity can initiate germinal vesicle (GV) breakdown in mammalian oocytes. It is crucial to maintain oocytes at the GV stage for a long period to analyze meiotic resumption in vitro. Meiotic resumption can be reversibly inhibited in isolated oocytes by cAMP modulator forskolin, cAMP analog dibutyryl cAMP (dbcAMP), or PDE inhibitors, milrinone (Mil), Cilostazol (CLZ), and 3-isobutyl-1-methylxanthine (IBMX). However, these chemicals negatively affect oocyte development and maturation when used independently. Here, we used ICR mice to develop a model that could maintain GV-stage arrest with minimal toxic effects on subsequent oocyte and embryonic development. We identified optimal concentrations of forskolin, dbcAMP, Mil, CLZ, IBMX, and their combinations for inhibiting oocyte meiotic resumption. Adverse effects were assessed according to subsequent development potential, including meiotic resumption after washout, first polar body extrusion, early apoptosis, double-strand DNA breaks, mitochondrial distribution, adenosine triphosphate levels, and embryonic development. Incubation with a combination of 50.0 µM dbcAMP and 10.0 µM IBMX efficiently inhibited meiotic resumption in GV-stage oocytes, with low toxicity on subsequent oocyte maturation and embryonic development. This work proposes a novel method with reduced toxicity to effectively arrest and maintain mouse oocytes at the GV stage.

High expression of type I inositol 1,4,5-trisphosphate receptor in the kidney of rats with hepatorenal syndrome.

AIM: To detect the expression of type I inositol 1,4,5-trisphosphate receptor (IP3RI) in the kidney of rats with hepatorenal syndrome (HRS). METHODS: One hundred and twenty-five Sprague-Dawley rats were randomly divided into four groups to receive an intravenous injection of D-galactosamine (D-GalN) plus lipopolysaccharide (LPS; group G/L, n = 50), D-GalN alone (group G, n = 25), LPS alone (group L, n = 25), and normal saline (group NS, n = 25), respectively. At 3, 6, 9, 12, and 24 h after injection, blood, liver, and kidney samples were collected. Hematoxylin-eosin staining of liver tissue was performed to assess hepatocyte necrosis. Electron microscopy was used to observe ultrastructural changes in the kidney. Western blot analysis and real-time PCR were performed to detect the expression of IP3RI protein and mRNA in the kidney, respectively. RESULTS: Hepatocyte necrosis was aggravated gradually, which was most significant at 12 h after treatment with D-galactosamine/lipopolysaccharide, and was characterized by massive hepatocyte necrosis. At the same time, serum levels of biochemical indicators including liver and kidney function indexes were all significantly changed. The structure of the renal glomerulus and tubules was normal at all time points. Western blot analysis indicated that IP3RI protein expression began to rise at 3 h (P < 0.05) and peaked at 12 h (P < 0.01). Real-time PCR demonstrated that IP3RI mRNA expression began to rise at 3 h (P < 0.05) and peaked at 9 h (P < 0.01). CONCLUSION: IP3RI protein expression is increased in the kidney of HRS rats, and may be regulated at the transcriptional level.

Interfering RNA against PKC-α inhibits TNF-α-induced IP3R1 expression and improves glomerular filtration rate in rats with fulminant hepatic failure.

We have reported that tumor necrosis factor-α (TNF-α) is critical for reduction of glomerular filtration rate (GFR) in rats with fulminant hepatic failure (FHF). The present study aims to evaluate the underlying mechanisms of decreased GFR during acute hepatic failure. Rats with FHF induced by d-galactosamine plus lipopolysaccharide (GalN/LPS) were injected intravenously with recombinant lentivirus harboring short hairpin RNA against the protein kinase C-α ( PKC-α) gene (Lenti-shRNA-PKC-α). GFR, serum levels of aminotransferases, creatinine, urea nitrogen, potassium, sodium, chloride, TNF-α, and endothelin-1 (ET-1), as well as type 1 inositol 1,4,5-trisphosphate receptor (IP3R1) expression in renal tissue were assessed. The effects of PKC-α silencing on TNF-α-induced IP3R1, specificity protein 1 (SP-1), and c-Jun NH2-terminal kinase (JNK) expression, as well as cytosolic calcium content were determined in glomerular mesangial cell (GMCs) with RNAi against PKC-α. Renal IP3R1 overexpression was abrogated by pre-treatment with Lenti-shRNA-PKC-α. The PKC-α silence significantly improved the compromised GFR, reduced Cr levels, and reversed the decrease in glomerular inulin space and the increase in glomerular calcium content in GalN/LPS-exposed rats. TNF-α treatment increased expression of PKC-α, IP3R1, specificity protein 1 (SP-1), JNK, and p-JNK in GMCs and increased Ca2 + release and binding activity of SP-1 to the IP3R1 promoter. These effects were blocked by transfection of siRNA against the PKC-α gene, and the PKC-α gene silence also restored cytosolic Ca2+ concentration. RNAi targeting PKC-α inhibited TNF-α-induced IP3R1 overexpression and in turn improved compromised GFR in the development of acute kidney injury during FHF in rats.

Development of a rat model of D-galactosamine/lipopolysaccharide induced hepatorenal syndrome.

AIM: To develop a practical and reproducible rat model of hepatorenal syndrome for further study of the pathophysiology of human hepatorenal syndrome. METHODS: Sprague-Dawley rats were intravenously injected with D-galactosamine and lipopolysaccharide (LPS) via the tail vein to induce fulminant hepatic failure to develop a model of hepatorenal syndrome. Liver and kidney function tests and plasma cytokine levels were measured after D-galactosamine/LPS administration, and hepatic and renal pathology was studied. Glomerular filtration rate was detected in conscious rats using micro-osmotic pump technology with fluorescein isothiocyanate-labelled inulin as a surrogate marker. RESULTS: Serum levels of biochemical indicators including liver and kidney function indexes and cytokines all significantly changed, especially at 12 h after D-galactosamine/LPS administration [alanine aminotransferase, 3389.5 ± 499.5 IU/L; blood urea nitrogen, 13.9 ± 1.3 mmol/L; Cr, 78.1 ± 2.9 µmol/L; K(+), 6.1 ± 0.5 mmol/L; Na(+), 130.9 ± 1.9 mmol/L; Cl(-), 90.2 ± 1.9 mmol/L; tumor necrosis factor-α, 1699.6 ± 599.1 pg/mL; endothelin-1, 95.9 ± 25.9 pg/mL; P < 0.05 compared with normal saline control group]. Hepatocyte necrosis was aggravated gradually, which was most significant at 12 h after treatment with D-galactosamine/LPS, and was characterized by massive hepatocyte necrosis, while the structures of glomeruli, proximal and distal tubules were normal. Glomerular filtration rate was significantly decreased to 30%-35% of the control group at 12 h after D-galactosamine/LPS administration [Glomerular filtration rate (GFR)1, 0.79 ± 0.11 mL/min; GFR2, 3.58 ± 0.49 mL/min·kgBW(-1); GFR3, 0.39 ± 0.99 mL/min·gKW(-1)]. The decreasing timing of GFR was consistent with that of the presence of hepatocyte necrosis and liver and kidney dysfunction. CONCLUSION: The joint use of D-galactosamine and LPS can induce liver and kidney dysfunction and decline of glomerular filtration rate in rats which is a successful rat model of hepatorenal syndrome.

Tumor necrosis factor-alpha-induced reduction of glomerular filtration rate in rats with fulminant hepatic failure.

The mechanism of renal failure during fulminant hepatic failure (FHF) or end-stage of liver disease is not fully understood. The present study aims to delineate the mechanisms of decreased glomerular filtration rate (GFR) in acute hepatic failure. A rat model of renal insufficiency in severe liver injury was established by lipopolysaccharide (LPS) plus D-galactosamine (GalN) exposure. GFR was evaluated by continuous infusion of fluorescein isothiocyanate-inulin with implanted micro-osmotic pumps. GalN/LPS intoxication resulted in severe hepatocyte toxicity as evidenced by liver histology and biochemical tests, whereas renal morphology remained normal. GFR was reduced by 33% of the controls 12 h after GalN/LPS exposure, accompanied with a decreased serum sodium levels, a marked increase in serum TNF-α and ET-1 levels as well as significantly upregulated renal type 1 inositol 1,4,5-trisphosphate receptor (IP3R1) expression. The upregulated IP3R1 expression was abrogated by the treatment of anti-TNF-α antibodies, but not by 2-aminoethoxydiphenylborate (2-APB), which blocks the inositol 1,4,5-trisphosphate signaling pathway. Treatments with either TNF-α antibodies or 2-APB also significantly improved the compromised GFR, elevated serum urea nitrogen and creatinine levels, and reversed the decrease in glomerular inulin space and the increase in glomerular calcium content in GalN/LPS-exposed rats. The extent of acute liver injury as reflected by serum ALT levels was much more attenuated by anti-TNF-α antibodies than by 2-APB. Liver histology further confirmed that anti-TNF-α antibodies conferred better protection than 2-APB in GalN/LPS-exposed rats. LPS-elicited TNF-α over-production is responsible for decreased GFR through IP3R1 overexpression, and the compromised GFR resulted in the development of acute renal failure in rats with FHF.