Development and evaluation of a direct TaqMan qPCR assay for the rapid detection of diverse carnivore amdoparvoviruses.

Amdoparvoviruses infect carnivore species, including mink, raccoon dog, fox, skunk, and red panda. Amdoparvovirus infection is a major cause of morbidity and mortality in farmed minks. Here, we developed a direct TaqMan qPCR assay for detection and quantification of carnivore amdoparvoviruses by using three primers and one probe based on the conserved VP2 gene. The detection limit for Aleutian mink disease virus (AMDV) and Raccoon dog and arctic fox amdoparvovirus (RFAV) were 4.06 × 101 copies/µl and 2.93 × 101 copies/µl, respectively. Both intra- and inter-assay variability were less than 2%. Among 74 carnivore samples, the positive rates for amdoparvoviruses were 62.2% (46/74) by direct TaqMan qPCR, while only 40.5% (30/74) by SYBR Green I qPCR. This result suggests that the direct TaqMan qPCR was more sensitive than the SYBR Green I qPCR. Additionally, the direct TaqMan qPCR is a rapid and sensitive method for liquid samples at microliter level as the assay employed the direct alkaline lysis method to obtain viral DNA and, therefore, eliminated the cumbersome steps in extracting DNA. Overall, the direct TaqMan qPCR assay possessed high specificity, sensitivity, and reproducibility, indicating that it can be used as a powerful tool for detection and quantification of various carnivore amdoparvoviruses in epidemiological and pathogenesis studies.

Focal adhesion kinase PTK2 autophosphorylation is not required for the activation of sodium-hydrogen exchange by decreased cell volume in the preimplantation mouse embryo.

SummaryRecovery from decreased cell volume is accomplished by a regulated increase of intracellular osmolarity. The acute response is activation of inorganic ion transport into the cell, the main effector of which is the Na+/H+ exchanger NHE1. NHE1 is rapidly activated by a cell volume decrease in early embryos, but how this occurs is incompletely understood. Elucidating cell volume-regulatory mechanisms in early embryos is important, as it has been shown that their dysregulation results in preimplantation developmental arrest. The kinase JAK2 has a role in volume-mediated NHE1 activation in at least some cells, including 2-cell stage mouse embryos. However, while 2-cell embryos show partial inhibition of NHE1 when JAK2 activity is blocked, NHE1 activation in 1-cell embryos is JAK2-independent, implying a requirement for additional signalling mechanisms. As focal adhesion kinase (FAK aka PTK2) becomes phosphorylated and activated in some cell types in response to decreased cell volume, we sought to determine whether it was involved in NHE1 activation in the early mouse embryo. FAK activity requires initial autophosphorylation of a tyrosine residue, Y397. However, FAK Y397 phosphorylation levels were not increased in either 1- or 2-cell embryos after cell volume was decreased. Furthermore, the selective FAK inhibitor PF-562271 did not affect NHE1 activation at concentrations that essentially eliminated Y397 phosphorylation. Thus, autophosphorylation of FAK Y397 does not appear to be required for NHE1 activation induced by a decrease in cell volume in early mouse embryos.

Identification of the JY-1 in sika deer and its effects on granulosa cells cultured in vitro.

The oocyte-specific protein JY-1 was reported as an important regulator of both function of ovarian granulosa cells and early embryogenesis in cattle. Here, we found that the transcripts of JY-1 were also present in sika deer granulosa cells (GCs) through in situ hybridization and qRT-PCR. The complete sika deer JY-1 coding sequence was identified, which had three exons separated by two introns. It was detected that JY-1 knockdown caused apoptosis and abnormal cell cycle progression in GCs of sika deer cultured in vitro. Taken together, these data suggest that JY-1 is involved in the regulation of proliferation in sika deer ovarian GCs.

Histone deacetylase inhibitor stimulates E2 and P4 secretion in sika deer ovarian granulosa cells at a moderate dose.

The histone deacetylase inhibitor (HDACi) and tumor suppressor play an important role in genome reorganization and epigenetic regulation. In this study, granulosa cells (GCs) isolated from sika deer ovaries were cultured and treated with different concentrations of trichostatin A (TSA) for 48 h. It was found that TSA inhibited GCs proliferation and induced GCs apoptosis by upregulating expression of BAX, meanwhile, downregulating expression of GLUT3, GLUT8, BCL-XL. In addition, TSA caused cell cycle arrest at the G1 and G2/M phase accompanied by reducing expression of Cyclin D2 and CDK4. TSA pretreatment increased DNMT3a, DNMT1, HDAC1, and HAT1 expression, and attenuated them when TAS higher than 50 nM. The protein levels of H3K9ac and H4K8ac in GCs were increased at 48 h after TSA treatment. TSA stimulated the secretion of estradiol and progesterone at a moderate dose. Our data suggest that TSA is important as a regulator of steroid hormone synthesis in granulosa cells during follicular development in the sika deer ovary.

Acute cell volume regulation by Janus kinase 2-mediated sodium/hydrogen exchange activation develops at the late one-cell stage in mouse preimplantation embryos.

Early preimplantation embryos are extremely sensitive to dysregulation of cell volume, which can lead to developmental arrest. It was previously shown that mouse embryos at the two-cell stage respond to a cell volume decrease by quickly activating Na+/H+ exchange via a signaling mechanism that involves the tyrosine kinase Janus kinase 2 (JAK2). However, it was not known whether this mechanism is active at the one-cell stage, when embryos are most sensitive to perturbed cell volume. Na+/H+ exchanger activity elicited by an induced cell volume decrease was significantly lower at the mid one-cell stage than at the late one-cell stage or during the two-cell stage. This activity could be completely blocked by the broad specificity tyrosine kinase inhibitor genistein at either stage, but only at the two-cell stage was there a substantial component of activity that was sensitive to low concentrations of the JAK2-selective inhibitors TG101348 or ruxolitinib. Western blots to detect active JAK2 phosphorylated on tyrosine Y1007/8 revealed that JAK2 became substantially phosphorylated in response to a cell volume decrease at the mid two-cell, but not mid one-cell stage. Such cell volume decrease-induced JAK2 phosphorylation appeared by the late one-cell stage. At least in part this appears to be due to an increase in total JAK2 protein at the late one-cell stage. Furthermore, TG101348 impaired maintenance of cell volume at the two-cell, but not mid one-cell, stages. Thus, cell volume homeostasis requiring Na+/H+ exchange signaled by JAK2 first becomes prominent during mouse embryonic development at the late one-cell stage.

Mitogen-activated protein kinase-activated protein kinase 2 is a critical regulator of pig oocyte meiotic maturation.

Mitogen-activated protein kinase (MAPK)-activated protein kinase 2 (MK2), a direct substrate of p38 MAPK, plays key roles in multiple cellular processes. In the present study, we showed that MK2 affected not only cumulus expansion, but also the oocyte meiotic cell cycle in porcine oocytes. Inhibition of MK2 arrested oocytes at the germinal vesicle (GV) stage or the prometaphase I/metaphase I stage. Unlike in mouse oocytes, where phosphorylated (p-) MK2 was localised at the minus end of spindle microtubules and close to the spindle poles, in porcine oocytes p-MK2 was concentrated at the spindle equator and localised at the plus end of spindle microtubules. Knockdown or inhibition of MK2 resulted in spindle defects: spindles were surrounded by irregular chromosome non-disjunction or by chromosomes detached from the spindles. MK2 regulated spindle organisation and chromosome alignment by connecting microtubules with kinetochores. In addition, unlike in mitotic cells and meiotic mouse oocytes, the MK2-p38 MAPK pathway may not play an important role during meiotic cell cycle in porcine oocytes. In conclusion, MK2 is an important regulator of porcine oocyte meiotic maturation.

Bi-directional communication with the cumulus cells is involved in the deficiency of XY oocytes in the components essential for proper second meiotic spindle assembly.

The oocyte becomes competent for embryonic development by involving mutual communication with cumulus cells (CCs) during folliculogenesis. How this communication takes place under physiological conditions is not fully understood. Current study examined oocyte-CCs communication in the XY sex-revered female mouse. We have previously found that the XY oocyte is defective in its cytoplasm, causing abnormal MII-spindle assembly and a failure in embryonic development. Our present study showed that transcript levels of Pfkp, Pkm2 and Ldh1 involved in glycolysis were lower in the CCs surrounding XY oocytes than in those surrounding XX oocytes. ATP contents in XY oocytes were also lower than those in XX oocytes, suggesting that lower glycolytic gene expression in CCs resulted in lower ATP contents in the enclosed oocyte. Co-culture of oocytectomized CC-oocyte complexes (COCs) with denuded oocytes showed that XY oocytes were less efficient than XX oocytes in promoting glycolytic gene expression in CCs. Furthermore, both glycolytic gene expression levels in CCs and ATP contents in oocytes of XY COCs increased to similar levels to those of XX COCs after culture for 20h in the presence of milrinone (=preincubation), which prevented spontaneous oocyte maturation. By increasing ATP levels in XY oocytes by either COC preincubation or ATP microinjection into oocytes prior to in vitro maturation, an improvement in MII-spindle assembly was observed. We conclude that the XY oocyte produces lesser amounts of paracrine factors that affect its companion CCs, which in turn make the ooplasm deficient in its components, including ATP, essential for MII-spindle assembly.

l-carnitine supplementation during vitrification of mouse germinal vesicle stage-oocytes and their subsequent in vitro maturation improves meiotic spindle configuration and mitochondrial distribution in metaphase II oocytes.

STUDY QUESTION: How does l-carnitine (LC) supplementation during vitrification and in vitro maturation (IVM) of germinal vesicle stage (GV)-oocytes improve the developmental competence of the resultant metaphase II (MII) oocytes? SUMMARY ANSWER: LC supplementation during both vitrification of GV-oocytes and their subsequent IVM improved nuclear maturation as well as meiotic spindle assembly and mitochondrial distribution in MII oocytes. WHAT IS KNOWN ALREADY: Vitrification of GV-oocytes results in a lower success rate of blastocyst development compared with non-vitrified oocytes. LC supplementation during both vitrification and IVM of mouse GV-oocytes significantly improves embryonic development after IVF. STUDY DESIGN, SIZE, DURATION: GV-oocytes were collected from (B6.DBA)F1 and B6 mouse strains and subjected to vitrification and warming with or without 3.72 mM LC supplementation. After IVM with or without LC supplementation, the rate of nuclear maturation and the quality of MII oocytes were evaluated. At least 20 oocytes/group were examined, and each experiment was repeated at least three times. All experiments were conducted during 2013-2014. PARTICIPANTS/MATERIALS, SETTING, METHODS: Extrusion of the first polar body in IVM oocytes was observed as an indication of nuclear maturation. Spindle assembly and chromosomal alignment were examined by immunostaining of α-tubulin and nuclear staining with 4,6-diamidino-2-phenylindole (DAPI). Mitochondrial distribution and oxidative activity were measured by staining with Mitotracker Green Fluorescence Mitochondria (Mitotracker Green FM) and chloromethyltetramethylrosamine (Mitotracker Orange CMTMRos), respectively. ATP levels were determined by using the Bioluminescent Somatic Cell Assay Kit. MAIN RESULTS AND THE ROLE OF CHANCE: LC supplementation during both vitrification and IVM of GV-oocytes significantly increased the proportions of oocytes with normal MII spindles to the levels comparable with those of non-vitrified oocytes in both mouse strains. While vitrification of GV-oocytes lowered the proportions of MII oocytes with peripherally concentrated mitochondrial distribution compared with non-vitrified oocytes, LC supplementation significantly increased the proportion of such oocytes in the (B6.DBA)F1 strain. LC supplementation decreased the proportion of oocytes with mitochondrial aggregates in both vitrified and non-vitrified oocytes in the B6 strain. The oxidative activity of mitochondria was mildly decreased by vitrification and drastically increased by LC supplementation irrespective of vitrification in both mouse strains. No change was found in ATP levels irrespective of vitrification or LC supplementation. Results were considered to be statistically significant at P < 0.05 by either χ(2)- or t-test. LIMITATIONS, REASONS FOR CAUTION: It remains to be tested whether beneficial effect of LC supplementation during vitrification and IVM of GV-oocytes leads to fetal development and birth of healthy offspring after embryo transfer to surrogate females. WIDER IMPLICATIONS OF THE FINDINGS: This protocol has the potential to improve the quality of vitrified human oocytes and embryos during assisted reproduction treatment. STUDY FUNDING/COMPETING INTEREST: Partially supported by the Natural Sciences and Engineering Research Council of Canada (NSERC) Discovery Grant and Mitacs Elevate Postdoctoral Fellowship, Canada.

Sodium fluoride disrupts DNA methylation of H19 and Peg3 imprinted genes during the early development of mouse embryo.

Sodium fluoride (NaF) is associated with embryonic and fetal development abnormalities, but the mechanism by which this occurs is unclear. DNA methylation, an important epigenetic reprogramming mechanism, is essential for normal embryonic development. Thus, we investigated the effect of NaF on DNA methylation in early mouse embryos, as well as mouse sperm and liver using bisulfite sequencing and ELISA. Data indicate that H19, a paternally imprinted gene, compared to control embryos, was less methylated in 8-cell embryos from pregnant mice treated with NaF (100 mg/l) in drinking water for 48 h. Peg3, a maternally imprinted gene, and the Line1 repeated sequence were similarly methylated in NaF-treated and control embryos. Oral ingestion of NaF for 35 days did not significantly change Line1 and genomic global DNA methylation in the liver. H19, Rasgrf1, Line1, and genomic global DNA methylation were also similar in NaF-treated and control sperm. Female mice mated with NaF-treated male mice (35 days) had less methylated H19, but Peg3 was significantly more methylated. Line1 was similarly methylated in treated 8-cell embryos, compared to control embryos. NaF treatment of male mice before copulation significantly increased the expression of H19 in blastocysts, whereas H19 expression was not detected in 8-cell embryos. Data suggest that NaF may interact directly with the embryo to disrupt the maintenance of normal gene imprinting during pregnancy. Long-term NaF exposure of males may not directly affect DNA methylation of the sperm and liver, but the sperm may signal to early embryos with abnormal gene imprinting.

L-carnitine supplementation during vitrification of mouse oocytes at the germinal vesicle stage improves preimplantation development following maturation and fertilization in vitro.

Oocyte cryopreservation is important for assisted reproductive technologies (ART). Although cryopreservation of metaphase II (MII) oocytes has been successfully used, MII oocytes are vulnerable to the damage inflicted by the freezing procedure. Cryopreservation of germinal vesicle stage oocytes (GV-oocytes) is an alternative choice; however, blastocyst development from GV-oocytes is limited largely due to the need for in vitro maturation (IVM). Herein, we evaluated the effects of l-carnitine (LC) supplementation during vitrification and thawing of mouse GV-oocytes, IVM, and embryo culture on preimplantation development after in vitro fertilization (IVF). We first compared the rate of embryonic development from the oocytes that had been collected at the GV stage from three mouse strains, (B6.DBA)F1, (B6.C3H)F1, and B6, and processed for IVM and IVF, as well as that from the oocytes matured in vivo, i.e. ovulated (IVO). Our results demonstrated that the rate of blastocyst development was the highest in the (B6.DBA)F1 strain and the lowest in the B6 strain. We then supplemented the IVM medium with 0.6 mg/ml LC. The rate of blastocyst development improved in the B6 but not in the (B6.DBA)F1 strain. Vitrification of GV-oocytes in the basic medium alone reduced the rate of blastocyst development in both of those mouse strains. LC supplementation to the IVM medium alone did not change the percentage of blastocyst development. However, LC supplementation to both vitrification and IVM media significantly improved blastocyst development to the levels comparable with those obtained from vitrified/thawed IVO oocytes in both of the (B6.DBA)F1 and B6 strains. We conclude that LC supplementation during vitrification is particularly efficient in improving the preimplantation development from the GV-oocytes that otherwise have lower developmental competence in culture.

Velvet antler water extract protects porcine oocytes from lipopolysaccharide-induced meiotic defects.

Previous studies have demonstrated that lipopolysaccharide (LPS), as a central toxic factor of gram-negative bacteria, can induce oxidative stress and cellular inflammation to result in the impairment of female fertility in different organisms. Particularly, it has harmful effects on the oocyte quality and subsequent embryonic development. However, the approach concerning how to prevent oocytes from LPS-induced deterioration still remains largely unexplored. We assessed the effective influences of velvet antler water extract (VAWE) by immunostaining and fluorescence intensity quantification on the meiotic maturation, mitochondrial function and sperm binding ability of oocytes under oxidative stress. Here, we report that VAWE treatment restores the quality of porcine oocytes exposed to LPS. Specifically, LPS exposure contributed to the failed oocyte maturation, reduced sperm binding ability and fertilization capability by disturbing the dynamics and arrangement of meiotic apparatuses and organelles, including spindle assembly, chromosome alignment, actin polymerization, mitochondrial dynamics and cortical granule distribution, the indicators of oocyte nuclear and cytoplasmic maturation. Notably, VAWE treatment recovered these meiotic defects by removing the LPS-induced excessive ROS and thus inhibiting the apoptosis. Collectively, our study illustrates that VAWE treatment is a feasible strategy to improve the oocyte quality deteriorated by the LPS-induced oxidative stress.

Involvement of testicular N-glycoproteome heterogeneity in seasonal spermatogenesis of the American mink (Neovison vison).

Spermatogenesis in the American mink is characterized by an annual cycle of transition involving completely inactive and fully activated stages. N-glycosylation of proteins has emerged as an important regulator as it affects protein folding, secretion, degradation, and activity. However, the function of protein N-glycosylation in seasonal spermatogenesis of the American mink remains unclear. In the present study, we established a proteome-wide stoichiometry of N-glycosylation in mink testes at various phases of spermatogenesis using N-linked glycosylated-peptide enrichment in combination with liquid chromatography-tandem mass spectrometry analysis. A total of 532 N-glycosylated sites matching the canonical Asn-X-Ser/Thr motif were identified in 357 testicular proteins. Both the number of glycoproteins and the sites of N-glycosylated proteins in mink testes were highly dynamic at different stages. Functional analyses showed that testicular proteins with different N-glycosylation might play a vital role in spermatogenesis by affecting their folding, distribution, stability, and activity. Overall, our data suggest that the dynamics of N-glycosylation of testicular proteins are involved in seasonal spermatogenesis in the American mink.

Glycine and Melatonin Improve Preimplantation Development of Porcine Oocytes Vitrified at the Germinal Vesicle Stage.

Lipid-rich porcine oocytes are extremely sensitive to cryopreservation compared to other low-lipid oocytes. Vitrification has outperformed slowing freezing in oocyte cryopreservation and is expected to improve further by minimizing cellular osmotic and/or oxidative stresses. In this study, we compared the effects of loading porcine cumulus-oocyte complexes with glycine (an organic osmolyte) or glycine plus melatonin (an endogenous antioxidant) during vitrification, thawing and subsequent maturation to mitigate osmotic injuries or osmotic and oxidative damages on the developmental potential of porcine oocytes. Our data demonstrated that glycine treatment significantly increased the vitrification efficiency of porcine oocytes to levels comparable to those observed with glycine plus melatonin treatment. It was manifested as the thawed oocyte viability, oocyte nuclear maturation, contents of reactive oxygen species, translocation of cortical granules and apoptotic occurrence in mature oocytes, levels of ATP and transcripts of glycolytic genes in cumulus cells (markers of oocyte quality), oocyte fertilization and blastocyst development. However, the latter was more likely than the former to increase ATP contents and normal mitochondrial distribution in mature oocytes. Taken together, our results suggest that mitigating osmotic and oxidative stresses induced by vitrification and thawing can further enhance the developmental competency of vitrified porcine oocytes at the germinal vesicle stage.

Phosphorylation of adducin-1 by TPX2 promotes interpolar microtubule homeostasis and precise chromosome segregation in mouse oocytes.

BACKGROUND: ADD1 (adducin-1) and TPX2 (targeting protein for Xklp2) are centrosomal proteins and regulate mitotic spindle assembly. Mammalian oocytes that segregate homologous chromosomes in Meiosis I and sister chromatids in Meiosis II with a spindle lacking centrosomes are more prone to chromosome segregation errors than in mitosis. However, the regulatory mechanisms of oocyte spindle assembly and the functions of ADD1 and TPX2 in this process remain elusive. RESULT: We found that the expression levels and localization of ADD1, S726 phosphorylated ADD1 (p-ADD1), and TPX2 proteins exhibited spindle assembly-dependent dynamic changes during mouse oocyte meiosis. Taxol treatment, which stabilizes the microtubule polymer and protects it from disassembly, made the signals of ADD1, p-ADD1, and TPX2 present in the microtubule organizing centers of small asters and spindles. Knockdown of approximately 60% of ADD1 protein levels destabilized interpolar microtubules in the meiotic spindle, resulting in aberrant chromosome alignment, reduced first polar body extrusion, and increased aneuploidy in metaphase II oocytes, but did not affect K-fiber homeostasis and the expression and localization of TPX2. Strikingly, TPX2 deficiency caused increased protein content of ADD1, but decreased expression and detachment of p-ADD1 from the spindle, thereby arresting mouse oocytes at the metaphase I stage with collapsed spindles. CONCLUSION: Phosphorylation of ADD1 at S726 by TPX2 mediates acentriolar spindle assembly and precise chromosome segregation in mouse oocytes.

Transcriptomic analysis of ovarian signaling at the emergence of the embryo from obligate diapause in the American mink (Neovison vison).

Mink embryonic diapause occurs when embryos, at the blastocyst stage, enter a state of a reversible arrest in development and metabolism. Some ovarian factors are required because ovariectomy leads to prevention of implantation in mink. Mechanisms regulating this process, however, remain largely unknown. To explore ovarian modifications associated with emergence of embryonic diapause in mink, there was comparison of transcriptomes after embryonic activation to when there was embryonic diapause using RNA-sequencing. A library of 655 differentially expressed genes (DEGs) of all assembled 33,656 genes was generated. Among these, 558 genes were annotated with 106 genes being expressed to a greater extent in ovaries during embryonic diapause, whereas 452 genes were more abundantly expressed in ovaries after embryonic activation. The major categories of genes with differential transcript abundances include metabolic pathways, metabolism of tryptophan, tyrosine and vitamin B6, oxidoreductase activity, calcium signaling pathway, steroid biosynthesis and lysosome. The APOE and APOA1 hub genes identified through the protein-protein interaction (PPI) analysis have important functions in cholesterol transport and steroidogenesis. Transcript abundances associated with 39 genes were investigated using RT-qPCR procedures to confirm RNA-sequencing data. Of 29 mRNA transcripts, 26 were validated using RNA-sequencing, whereas three of ten indistinguishable genes determined using RNA-sequencing were confirmed. Most of these verified DEGs are involved in the prolactin signaling pathway, formation of functional corpora lutea, and steroid synthesis, suggesting these biological processes are implicated in embryonic reactivation. Overall, results provide new insights into ovarian signaling at the time of emergence of the blastocyst from diapause in mink.

Changes in estrogen receptor-alpha variant (ER-alpha36) expression during mouse ovary development and oocyte meiotic maturation.

The biological effects of estrogens are largely mediated through estrogen receptors (ERs), which belong to the nuclear receptor gene family of transcription factors. ER-alpha36 has been recently identified as a new variant of ERalpha, but its expression and roles in female reproduction system remain unknown. Immunocytochemistry and confocal microscopy were employed to observe ER-alpha36 distribution in mouse ovary during postnatal development and in oocyte during meiotic maturation. ER-alpha36 was consistently present in the nuclei of oocytes regardless of follicular growth stage and mouse age until germinal vesicle breakdown (GVBD). Its immunosignal was smeared in granulosa cells. However, the ER-alpha36 signal is up-regulated and found in cytoplasm with little or no nuclear staining during corpus luteum development. ER-alpha36 was also found in theca cells. We showed by Western blot that ER-alpha36 was expressed in mouse oocytes at various maturation stages. When the function of nuclear ER-alpha36 was blocked by microinjecting anti-ER-alpha36 specific antibody into the germinal vesicle (GV) of mouse oocytes, the first polar body emission occurred earlier in a higher proportion of oocytes compared to the control. These results suggest that ER-alpha36 may play critical roles in mouse ovarian folliculogenesis and oocyte development.

Comparative Transcriptome Analysis of Unusual Localized Skin Laxity in Sika Deer (Cervus nippon).

Cutis laxa represents a heterogeneous group of rare, inherited, or acquired connective tissue disorders with the common feature of loose and redundant skin with decreased elasticity. The skin of affected deer showed abnormal collagen fiber morphology. To identify the differentially expressed genes of the unusual localized skin laxity in sika deer, we performed transcriptome analysis in the affected and control sika deer. The transcriptome analysis showed 700 genes with significant differential expression in the affected skin as compared with normal skin. Pathway analysis revealed an enrichment of genes involved in tumor necrosis factor signaling, the extracellular matrix-receptor interaction, platelet activation, and Huntington's disease. A gene network was constructed, and the hub nodes such as PTGS2, THBS1, COL1A1, FOS, and NOS3 were found through PPI network analysis, which may contributed to the unusual localized skin laxity in sika deer. Abnormal expression patterns of genes during the development of the affected sika deer were successfully uncovered in the present study, which provides a reference for revealing the related mechanism underlying cutis laxa in sika deer and human beings.

Heat stress causes aberrant DNA methylation of H19 and Igf-2r in mouse blastocysts.

To gain a better understanding of the methylation imprinting changes associated with heat stress in early development, we used bisulfite sequencing and bisulfite restriction analysis to examine the DNA methylation status of imprinted genes in early embryos (blastocysts). The paternal imprinted genes, H19 and Igf-2r, had lower methylation levels in heat-stressed embryos than in control embryos, whereas the maternal imprinted genes, Peg3 and Peg1, had similar methylation pattern in heat-stressed embryos and in control embryos. Our results indicate that heat stress may induce aberrant methylation imprinting, which results in developmental failure of mouse embryos, and that the effects of heat shock on methylation imprinting may be gene-specific.

PI3-kinase and mitogen-activated protein kinase in cumulus cells mediate EGF-induced meiotic resumption of porcine oocyte.

Previous studies have shown that epidermal growth factor (EGF) has the ability to promote in vitro cultured porcine oocyte maturation. However, little is known about the detailed downstream events in EGF-induced meiotic resumption. We designed this study to determine the relationship of EGF, EGFR, phosphatidylinositol 3-kinase (PI3-kinase), MAPK, and germinal vesicle breakdown (GVBD) during oocyte maturation. Our results showed that GVBD in cumulus-enclosed oocytes (CEOs) but not in denuded oocytes (DOs) was induced by EGF in a dose-dependent manner, which indicated that cumulus cells but not oocyte itself were the main target for EGF-induced meiotic resumption. Furthermore, we found that MAPK in cumulus cells rather than in oocyte was activated immediately after EGF administration. To explore whether EGF exerts its functions through MAPK pathway, the activities of EGF receptor (EGFR) and MAPK were inhibited by employing AG1478 and U0126, respectively. Inhibition of MAPK blocked EGF-induced GVBD, whereas inhibition of EGFR prevented MAPK activation. Both AG1478 and U0126 could lead to the failure of EGF-induced GVBD singly. Notably, we found that LY294002, a specific inhibitor of PI3-kinase, effectively inhibited EGF-induced MAPK activation as well as subsequent oocyte meiotic resumption and this inhibition could not be reversed by adding additional EGF. Thus, PI3-kinase-induced MAPK activation in cumulus cells mediated EGF-induced meiotic resumption in porcine CEOs. Together, this study provides evidences demonstrating a linear relationship of EGF/EGFR, PI3-kinase, MAPK and GVBD and presents a relatively definitive mechanism of EGF-induced meiotic resumption of porcine oocyte.