Locational and functional characterization of PI4KB in the mouse embryo.

Phosphatidylinositol 4-kinase beta (PI4KB) is a member of the PI4K family, which is mainly enriched and functions in the Golgi apparatus. The kinase domain of PI4KB catalyzes the phosphorylation of phosphatidylinositol to form phosphatidylinositol 4-phosphate, a process that regulates various sub-cellular events, such as non-vesicular cholesterol and ceramide transport, protein glycosylation, and vesicle transport, as well as cytoplasmic division. In this study, a strain of PI4KB knockout mouse, immunofluorescence, reverse transcription polymerase chain reaction and microinjection were used to characterize the cytological location and biological function of PI4KB in the mouse embryos. we found that knocking down Pi4kb in mouse embryos resulted in embryonic lethality at around embryonic day (E) 7.5. Additionally, we observed dramatic fluctuations in PI4KB expression during the development of preimplantation embryos, with high expression in the 4-cell and morula stages. PI4KB colocalized with the Golgi marker protein TGN46 in the perinuclear and cytoplasmic regions in early blastomeres. Postimplantation, PI4KB was highly expressed in the epiblast of E7.5 embryos. Treatment of embryos with PI4KB inhibitors was found to inhibit the development of the morula into a blastocyst and the normal progression of cytoplasmic division during the formation of a 4-cell embryo. These findings suggest that PI4KB plays an important role in mouse embryogenesis by regulating various intracellular vital functions of embryonic cells.

Endoplasmic reticulum stress, a novel significant mechanism responsible for DEHP-induced increased distance between seminiferous tubule of mouse testis.

Di(2-ethylhexyl) phthalate (DEHP), a widely existed endocrine disruptor, has been concerned for many years owing to its toxicity in male reproductive development. In this study, we investigated the reproductive effects and the mechanism of mouse testis after in uterus exposure to the plasticizer DEHP. We found that the UPR signaling pathway could be fully activated after DEHP treatment. In uterus DEHP exposure significantly increased abnormal morphology seminiferous tubules, expanded the distance between the tubules as well as caused abnormal endoplasmic reticulum (ER) ultrastructure, which could be reversed by 4-phenylbutyrate (4-PBA), an ER stress inhibitor. In addition, DEHP-induced ER stress pathway promoted a decline in protein expression, including cadherin protein N-cadherin in testis, which could also be reversed by 4-PBA. Taken together, our results provide compelling evidence that the ER stress would be a novel significant mechanism responsible for DEHP-induced the increased the distance between seminiferous tubule by reducing the N-cadherin expression.

Effect of Acrylamide on Oocyte Nuclear Maturation and Cumulus Cells Apoptosis in Mouse In Vitro.

Acrylamide (ACR) is a chemical compound with severe neurotoxicity, genotoxicity, carcinogenicity and reproductive toxicity. Recent studies showed that ACR impairs the function of reproductive organs, e.g., epididymis and testes. In vitro maturation of mouse oocyte is a sensitive assay to identify potential chemical hazard to female fertility. The aim of this study was to evaluate the adverse effects of ACR on the nuclear maturation and cumulus cells apoptosis of mouse oocytes in vitro. Cumulus-oocyte complexes were incubated in a maturation medium containing 0, 5, 10 and 20 µM of ACR. Chromosome alignment and spindle morphology of oocytes was determined by immunofluorescence and confocal microscopy. Our results showed that oocytes exposed to different doses of ACR in vitro were associated with a significant decrease of oocyte maturation, significant increase of chromosome misalignment rate, occurrence of abnormal spindle configurations, and the inhibition of oocyte parthenogenetic activation. Furthermore, apoptosis of cumulus cells was determined by TUNEL and CASPASE-3 assay. Results showed that apoptosis in cumulus cells was enhanced and the expression of CASPASE-3 was increased after cumulus-oocyte complexes were exposed to ACR. Therefore, ACR may affect the nuclear maturation of oocytes via the apoptosis of cumulus cells in vitro.

[Effects of electroacupuncture on embryo implanted potential for patients with infertility of different symptom complex].

OBJECTIVE: To explore the impacts of electroacupuncture on embryo implanted potential and its molecular mechanism in the patients with infertility of different symptom complex. METHODS: Among the patients with infertility treated with electroacupuncture and in vitro fertilization and embryo transplantation (IVF-ET), 82 cases of kidney deficiency (group A), 74 cases of liver qi stagnation (group B) and 54 cases of phlegm dampness (group C) were selected. All of the patients in three groups received long-program ovarian hyper-stimulation. Additionally, electroacupuncturecan was applied before controlled ovarian hyper-stimulation (COH) and in the process of ovarian hyper-stimulation. The levels of human leukocyte antigen-G (HLA-G) in the serum were determined on the 2nd day of the menstruation (M2), on the day of human Chorionic Gonadotropin (hCG) injection and on the day of embryo transplantation in the culture solution in three groups separately. The fertilization rate, implantation rate and clinical pregnancy rate were observed for the patients in three groups. RESULTS: The levels of HLA-G in the serum on hCG injection day and in the culture solution on embryo transplantation day in group A and B were significantly higher than those in group C (all P < 0.05). But, there was no significant difference in serum HLA-G levels on M2 day among three groups. The high-quality embryo rate in either group A (73.6%, 352/478) or group B (70.6%, 379/537) was higher significantly than that in group C (54.2%, 208/384) separately, presenting statistical significant difference (all P < 0.01). But there were no significant differences in clinical pregnancy rate, fertilization rate and cleavage rate among three groups. CONCLUSION: Electroacupuncture can increase the contents of HLA-G in the body and the level of HLA-G secreted in embryos for the patients in the process of IVF-ET. Eventually, the pregnancy outcome and the pregnancy rate are improved. The clinical effects of electroacupuncture for the patients of kidney deficiency and liver qi stagnation are better than those for the patients of phlegm dampness.

Transplantation of male pronucleus derived from in vitro fertilization of enucleated oocyte into parthenogenetically activated oocyte results in live offspring in mouse.

In this study, inter-strain reconstructed embryos were produced by combining the female pronucleus of Kunming mouse (white) with male pronucleus of C57BL/6 strain (black). Metaphase II (MII) oocytes of Kunming mouse were enucleated and the zona pellucida was removed. Then, the enucleated oocytes were inseminated by capacitated sperm of C57BL/6 mouse in vitro. At the same time, MII oocytes of Kunming mouse were artificially activated using strontium chloride solution, which did not contain cytochalasin B. Finally, we removed the male pronucleus derived from C57BL/6 sperm and injected it into a parthenogenetically activated one-pronucleus oocyte by micromanipulation. The reconstructed 2-cell embryos were transplanted into the oviducts of 22 foster mother mice, each receiving about 20 embryos. In the end, seven healthy and live pups were born from one recipient.