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.

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.

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.

p38α MAPK is a MTOC-associated protein regulating spindle assembly, spindle length and accurate chromosome segregation during mouse oocyte meiotic maturation.

P38αMAPK (p38α) is usually activated in response to various stresses and plays a role in the inhibition of cell proliferation and tumor progression, but little is known about its roles in meiotic spindle assembly. In this study, we characterized the dynamic localization of p38α and explored its function in mouse oocyte meiotic maturation. P38α specifically colocalized with γ-tubulin and Plk1 at the center of MTOCs and spindle poles. Depletion of p38α by specific morpholino injection resulted in severely defective spindles and misaligned chromosomes probably via MK2 dephosphorylation. Notably, depletion of p38α led to significant spindle pole defects, spindle elongation, non-tethered kinetochore microtubules and increased microtubule tension. The disruption of spindle stability was coupled with decreased γ-tubulin and Plk1 at MTOCs. Overexpression of Eg5, a conserved motor protein, also caused spindle elongation and its morpholino injection almost completely rescued spindle elongation caused by p38α depletion. In addition, p38α-depletion decreased BubR1 and interfered with spindle assembly checkpoint (SAC), which resulted in aneuploid oocytes. Together, these data indicate that p38α is an important component of MTOCs, which regulates spindle assembly and spindle length, as well as stabilizes the spindle and spindle poles. Perturbed SAC and abnormal microtubule tension may be responsible for the misaligned chromosomes and high aneuploidy in p38α-depleted mouse oocytes.

MAPK-activated protein kinase 2 is required for mouse meiotic spindle assembly and kinetochore-microtubule attachment.

MAPK-activated protein kinase 2 (MK2), a direct substrate of p38 MAPK, plays key roles in multiple physiological functions in mitosis. Here, we show for the first time the unique distribution pattern of MK2 in meiosis. Phospho-MK2 was localized on bipolar spindle minus ends and along the interstitial axes of homologous chromosomes extending over centromere regions and arm regions at metaphase of first meiosis (MI stage) in mouse oocytes. At metaphase of second meiosis (MII stage), p-MK2 was localized on the bipolar spindle minus ends and at the inner centromere region of sister chromatids as dots. Knockdown or inhibition of MK2 resulted in spindle defects. Spindles were surrounded by irregular nondisjunction chromosomes, which were arranged in an amphitelic or syntelic/monotelic manner, or chromosomes detached from the spindles. Kinetochore-microtubule attachments were impaired in MK2-deficient oocytes because spindle microtubules became unstable in response to cold treatment. In addition, homologous chromosome segregation and meiosis progression were inhibited in these oocytes. Our data suggest that MK2 may be essential for functional meiotic bipolar spindle formation, chromosome segregation and proper kinetochore-microtubule attachments.

Effect of gonadotropins on oocyte maturation in vitro: an animal model.

OBJECTIVE: Analysis of the effects of human-derived gonadotropin drugs, FSH and LH (Repronex) and hCG (Novarel), on oocyte maturation, using a porcine oocyte in vitro maturation system as a culture model. DESIGN: Randomized research experimental study. SETTING: Academic basic research laboratory. ANIMALS: Prepubertal gilts that were slaughtered in the local slaughter house. INTERVENTION(S): Oocytes will be exposed to immunofluorescent staining and confocal laser scanning microscopy: Western blot analysis on cumulus-oocyte-complexes following treatment with different concentrations of the gonadotropin drugs Repronex, Novarel, and a Repronex and Novarel combination. MAIN OUTCOME MEASURE(S): Analysis of porcine oocyte spindle and chromosomal configuration with alpha-tubulin-fluorescein isothiocyanate antibody and propidium iodide staining. Porcine oocyte mitochondrial distribution and aggregation pattern staining was assessed with Mito Tracker Red CMXRox probe. Porcine oocyte cortical granule distribution was observed via peanut agglutinin-fluorescein isothiocyannate staining; Western blot analysis detected extra-cellular signal-regulated kinase 1/2 activation in cumulus cells. RESULT(S): An increase of gonadotropin concentration in the culture medium resulted in an increase in the following: the percentage of oocytes reaching metaphase II, normal configuration of the spindle, normal chromosomal alignment, cortical granule migration, and mitochondrial aggregation. Levels of nuclear and cytoplasmic maturation peaked as the concentration of gonadotropins approached its threshold level. CONCLUSION(S): Addition of a threshold concentration of the gonadotropin drugs Repronex, Novarel, and a combination of the two can significantly improve porcine oocyte maturation in vitro.

Perturbation of survivin expression affects chromosome alignment and spindle checkpoint in mouse oocyte meiotic maturation.

Survivin is a member of inhibitors of apoptosis proteins (IAPs), which have multiple regulatory functions in mitosis, but its roles in meiosis remain unknown. Here, we report its expression, localization and functions in mouse oocyte meiosis. Survivin displayed a maximal expression level in GV stage, and then gradually decreased from Pro-MI to MII stages. Immunofluorescent staining showed that survivin was restricted to the germinal vesicle, associated with centromeres from pro-metaphase I to metaphase I stages, distributed at the midzone and midbody of anaphase and telophase spindles, and located to centromeres at metaphase II stages. Depletion of survivin by antibody injection and morpholino injection resulted in severe chromosome misalignment, precocious polar body extrusion, and larger-than-normal polar bodies. Overexpression of survivin resulted in severe chromosome misalignment and prometaphase I or metaphase I arrest in a large proportion of oocytes. Our data suggest that survivin is required for chromosome alignment and that it may regulate spindle checkpoint activity during mouse oocyte meiosis.

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.

Involvement of calcium/calmodulin-dependent protein kinase kinase in meiotic maturation of pig oocytes.

Calcium (Ca(2+))/calmodulin-dependent protein kinase kinase (CaMKK) is a novel member of Ca(2+)/calmodulin-dependent protein kinase (CaMK) family, whose physiological roles in regulating meiotic cell cycle needs to be determined. We showed by Western blot that CaMKK was expressed in pig oocytes at various maturation stages. Confocal microscopy was employed to observe CaMKK distribution. In oocytes at the germinal vesicle (GV) or prometaphase I (pro-MI) stage, CaMKK was distributed in the nucleus, around the condensed chromatin and the cortex of the cell. At metaphase I (MI) stage, CaMKK was concentrated in the cortex of the cell. After transition to anaphase I or telophase I stage, CaMKK was detected around the separating chromosomes and in the cortex of the cell. At metaphase II (MII) stage, CaMKK was localized to the cortex of the cell, with a thicker area near the first polar body (PB1). Treatment of pig cumulus-enclosed oocytes with STO-609, a membrane-permeable CaMKK inhibitor, resulted in the delay/inhibition of the meiotic resumption and the inhibition of first polar body emission. The correlation between CaMKK and microfilaments during meiotic maturation of pig oocytes was then studied. CaMKK and microfilaments were colocalized from MI to MII during porcine oocyte maturation. After oocytes were treated with STO-609, microfilaments were depolymerized, while in oocytes exposed to cytochalasin B (CB), a microfilament polymerization inhibitor, CaMKK became diffused evenly throughout the cell. These data suggest that CaMKK is involved in regulating the meiotic cell cycle probably by interacting with microfilaments in pig oocytes.

Testosterone potentially triggers meiotic resumption by activation of intra-oocyte SRC and MAPK in porcine oocytes.

The role of androgen and androgen receptors (ARs) in males has been well established. This steroid and its receptor also exist in follicles, but their functions are still unclear. In this study, using a culture system containing a low dose of hypoxanthine, we revealed the positive contribution of testosterone to oocyte meiotic resumption. By performing ultracentrifugation to allow clear visualization of porcine germinal vesicles, our results provide evidence that mitogen-activated protein kinase (MAPK) in the oocyte itself but not in cumulus cells was activated before germinal vesicle breakdown (GVBD) after testosterone treatment. We further explored the signal cascade of testosterone-triggered GVBD and showed significant contributions of AR to testosterone-induced MAPK activation and GVBD. By using a potent and selective inhibitor of SRC and detecting activation of the kinase, we found that testosterone activated SRC in oocytes but not in cumulus cells and that SRC (as an essential upstream molecule of MAPK) mediated this testosterone- and AR-promoted reinitiation of meiosis. The present findings propose an undefined signaling pathway and suggest the potential competence of testosterone for meiotic resumption in mammalian oocytes.

Involvement of Polo-like kinase 1 in MEK1/2-regulated spindle formation during mouse oocyte meiosis.

Our recent studies have shown that MEK1/2 is a critical regulator of microtubule organization and spindle formation during oocyte meiosis. In the present study, we found that Plk1 colocalized with p-MEK1/2 at various meiotic stages after GVBD when microtubule began to organize. Also, Plk1 was able to coimmunoprecipitate with p-MEK1/2 in metaphase I stage mouse oocyte extracts, further confirming their physical interaction. Taxol-treated oocytes exhibited a number of cytoplasmic asters, in which both Plk1 and p-MEK1/2 were present, indicating that they might be complexed to participate in the acentrosomal spindle formation at the MTOCs during oocyte meiosis. Depolymerization of microtubules by nocodazole resulted in the complete disassembly of spindles, but Plk1 remained associated with p-MEK1/2, accumulating in the vicinity of chromosomes. More importantly, when p-MEK1/2 activity was blocked by U0126, Plk1 lost its normal localization at the spindle poles, which might be one of the most vital factors causing the abnormal spindles in MEK1/2-inhibited oocytes. Taken together, these data indicate that Plk1 and MEK1/2 regulate the spindle formation in the same pathway and that Plk1 is involved in MEK1/2-regulated spindle assembly during mouse oocyte meiotic maturation.

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.