Cadmium Exposure of Female Mice Impairs the Meiotic Maturation of Oocytes and Subsequent Embryonic Development.

Cadmium is one major pollutant that is highly toxic to animals and humans. The mechanism of cadmium toxicity on the female reproductive system, particularly oocyte maturation and fertility, remains to be clarified. In this study, we used a mouse model to investigate the effects of cadmium in the drinking water on the meiotic maturation of oocytes and subsequent embryonic development, and the underlying mechanisms associated with the impairment of oocyte maturation such as mitochondrial distribution and histone modifications. Our results show that cadmium exposure decreased the number of ovulated oocytes and impaired oocyte meiotic maturation rate both in vivo and in vitro. The embryonic development after fertilization was also impaired even when the potential hazards of cadmium on the spermatozoa or the genital tract have been excluded by fertilization and embryonic development in culture. Cadmium exposure disrupted meiotic spindle morphology and actin filament, which are responsible for successful chromosome segregation and the polar body extrusion during oocyte maturation and fertilization. ATP contents, which are required for proper meiotic spindle assembly in the oocyte, were decreased, consistent with altered mitochondrial distribution after cadmium exposure. Finally, cadmium exposure affected the levels of H3K9me2 and H4K12ac in the oocyte, which are closely associated with the acquisition of oocyte developmental competence and subsequent embryonic development. In conclusion, cadmium exposure in female mice impaired meiotic maturation of oocytes and subsequent embryonic development by affecting the cytoskeletal organization, mitochondrial function, and histone modifications.

A lack of coordination between sister-chromatids segregation and cytokinesis in the oocytes of B6.YTIR (XY) sex-reversed female mice.

The B6.YTIR (XY) mouse develops bilateral ovaries despite the expression of the testis-determining gene Sry during gonadal differentiation. We reported that the oocytes of the XY female are defective in their cytoplasm, resulting in a failure in the second meiotic division after activation or fertilization in vitro. However, the mechanism of meiotic failure or the cause of infertility remained to be clarified. In the present study, we obtained mature oocytes from XY females by superovulation and confirmed that these oocytes also fail in zygotic development. By using confocal microscopy 3D-analysis, we demonstrated that meiotic spindles were properly positioned and oriented in the MII-oocytes from XY females. After parthenogenic activation, fewer oocytes from XY females extruded the second polar body, and in those oocytes, sister-chromatids were often separated but neither set entered the second polar body. ARP2, F-actin, and ORC4, known to play roles in asymmetric meiotic division, were initially localized along the ooplasmic membrane and concentrated over the MII-spindle but lost their cortical polarity after activation while the sister-chromatids moved away from the oolemma in the oocytes from XY females. Our results indicate that the second polar body extrusion is uncoupled from the sister-chromatids separation in the oocytes from XY female mouse.

Cadmium-induced autophagy is mediated by oxidative signaling in PC-12 cells and is associated with cytoprotection.

Oxidative stress induced by cadmium (Cd) is a common phenomenon that has been observed in numerous studies. However, the underlying mechanism remains unknown. Recently, exposure of PC-12 cells to Cd has been shown to activate autophagy, which acts as a temporary survival pathway under stressful conditions by delaying the occurrence of apoptosis. The present study investigated the impact of oxidative stress on Cd­induced autophagy in PC-12 cells. The results demonstrated that Cd­induced autophagy (following treatment with Cd for 4 h), increased the levels of intracellular reactive oxygen species (ROS), decreased the mitochondrial membrane potential and resulted in apoptosis. A treatment with chloroquine (CQ; an autophagic inhibitor) sensitized the PC­12 cells to Cd, due to the increased production of ROS, which was associated with the incapacity to reduce mitochondrial and cell death. N-acetyl-L-cysteine, an antioxidant agent, decreased Cd-induced autophagy and reduced intracellular ROS levels, but enhanced CQ­induced apoptotic cell death. These findings indicate that moderate levels of ROS are essential in the regulation of Cd-induced autophagy, which subsequently enhances cell survival. Thus, the results of the present study provide an insight for future investigation of Cd-induced neurotoxicity.

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.

Demethylation of LHR in dehydroepiandrosterone-induced mouse model of polycystic ovary syndrome.

The cause of polycystic ovary syndrome (PCOS), a complex endocrine disorder, is unknown, but its familial aggregation implies underlying genetic influences. Hyperandrogenemia is regarded as a major endocrine character of the PCOS. In this study, we employed bisulfite sequencing and bisulfite restriction analysis to investigate the DNA methylation status of LHR, AR, FSHR and H19 in dehydroepiandrosterone (DHEA)-induced mouse PCOS model. The result showed that methylation of LHR was lost in ovary from induced PCOS mouse. However, AR, FSHR and H19 had similar methylation pattern in DHEA-treated group and control groups. These data provide evidence for close linkage between DNA demethylation of LHR and PCOS.

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.

Loss of methylation imprint of Snrpn in postovulatory aging mouse oocyte.

Prolonged residence of postovulatory oocyte in the oviduct or prolonged culture in vitro can lead to oocyte aging, which significantly affects pre- and post-implantation embryo development. In this study, we employed bisulfite sequencing and COBRA methods to investigate the DNA methylation status of differentially methylated regions (DMRs) of Snrpn and Peg1/Mest, two maternally imprinted genes, in postovulatory oocytes aged in vivo and in vitro. The results showed that Snrpn DMR was clearly demethylated in oocytes aged in vivo at 29h post-hCG and in denuded oocytes aged in vitro for the same time period. However, Peg1/Mest did not show any demethylation in all aged groups at 29h post-hCG. These data indicate that oocytes undergo time-dependent demethylation of Snrpn DMR during the process of postovulatory aging.

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.

Diploid parthenogenetic embryos adopt a maternal-type methylation pattern on both sets of maternal chromosomes.

Epigenetic modifications are closely associated with embryo developmental potential. One of the epigenetic modifications thought to be involved in genomic imprinting is DNA methylation. Here we show that the maternally imprinted genes Snrpn and Peg1/Mest were nearly unmethylated or heavily methylated, respectively, in their differentially methylated regions (DMRs) at the two-cell stage in parthenogenetic embryos. However, both genes were gradually de novo methylated, with almost complete methylation of all CpG sites by the morula stage in parthenogenetic embryos. Unexpectedly, another maternally imprinted gene, Peg3, showed distinct dynamics of methylation during preimplantation development of diploid parthenogenetic embryos. Peg3 showed seemingly normal methylation patterns at the two-cell and morula stages, but was also strongly de novo methylated in parthenogenetic blastocysts. In contrast, the paternally imprinted genes H19 and Rasgrf1 showed complete unmethylation of their DMRs at the morula stage in parthenogenetic embryos. These results indicate that diploid parthenogenetic embryos adopt a maternal-type methylation pattern on both sets of maternal chromosomes and that the aberrantly homogeneous status of methylation imprints may partially account for developmental failure.