Obesity was shown to cause reproductive dysfunctions such as reduced conception, infertility and early pregnancy loss. However, the possible effects of obesity on oocyte quality are still not fully understood. In this study we investigated the effects of both diet and gene mutation induced obesity on impairments in mouse oocyte polarization, oxidative stress, and epigenetic modifications. Our results showed that high-fat diet induced obesity (HFD) and gene mutation induced obesity (ob/ob) could both impair oocyte meiotic maturation, disrupt spindle morphology, and reduce oocyte polarity. Oocytes from obese mice underwent oxidative stress, as shown by high DHE and ROS levels. Abnormal mitochondrial distributions and structures were observed in oocytes from obese groups of mice and early apoptosis signals were detected, which suggesting that oxidative stress had impaired mitochondrial function and resulted in oocyte apoptosis. Our results also showed that 5 mC levels and H3K9 and H3K27 methylation levels were altered in oocytes from obese mice, which indicated that DNA methylation and histone methylation had been affected. Our results showed that both HFD and ob/ob induced obesity affected oocyte maturation and that oxidative stress-induced early apoptosis and altered epigenetic modifications may be the reasons for reduced oocyte quality in obese mice.

Mycotoxins which mainly consist of Aflatoxin (AF), Zearalenone (ZEN) and Deoxynivalenol (DON) are commonly found in many food commodities. Although each component has been shown to cause liver toxicity and oxidative stress in several species, there is no evidence regarding the effect of naturally contained multiple mycotoxins on tissue toxicity and oxidative stress in vivo. In the present study, mycotoxins-contaminated maize (AF 597 µg/kg, ZEN 729 µg/kg, DON 3.1 mg/kg maize) was incorporated into the diet at three different doses (0, 5 and 20%) to feed the mice, and blood and tissue samples were collected to examine the oxidative stress related indexes. The results showed that the indexes of liver, kidney and spleen were all increased and the liver and kidney morphologies changed in the mycotoxin-treated mice. Also, the treatment resulted in the elevated glutathione peroxidase (GPx) activity and malondialdehyde (MDA) level in the serum and liver, indicating the presence of the oxidative stress. Moreover, the decrease of catalase (CAT) activity in the serum, liver and kidney as well as superoxide dismutase (SOD) activity in the liver and kidney tissue further confirmed the occurrence of oxidative stress. In conclusion, our data indicate that the naturally contained mycotoxins are toxic in vivo and able to induce the oxidant stress in the mouse.

Abstract. Mitogen-activated protein kinase (MAPK) and maturation/M phase promoting factor (MPF) play crucial roles in oocyte meiotic maturation in mammals. However, the underlying molecular mechanisms have not been addressed. In this study, the effects of the MEK/MAPK pathway inhibitor U0126 and the MPF inhibitor roscovitine on meiotic maturation and maternal gene expression in pig cumulus-oocyte complexes (COCs) and denuded oocytes (DOs) were investigated. Both inhibitors can reversibly block the resumption of meiosis in pig oocytes. COCs or DOs initially cultured in drug-free medium for 24 h and then transferred to medium containing U0126 showed normal progress to the Metaphase II stage (MII); (90.38 vs. 92.16% control group). In contrast, roscovitine treatment from 24 to 44 h significantly inhibited maturation of COCs and DOs. To explore the underlying molecular mechanisms, expression patterns and polyadenylation states of five representative maternal transcripts, cyclin B1, Cdc2, C-mos, GDF9 and BMP15, were examined by real-time PCR and poly(A)-test PCR (PAT assay). U0126 treatment resulted in aberrant expression of Cdc2 and GDF9, while roscovitine significantly maintained all five maternal transcripts at very high levels in treated COCs compared with the control group. The polyadenylation of these mRNAs increased as well. Furthermore, the experiments were repeated in DOs, and the results also indicated that both Cdc2 and GDF9 showed significantly higher expression in both mRNA and polyadenylation levels in the drug treatment groups. Together, these results provide the first demonstration in a mammalian system that MAPK and MPF play important roles in regulation of maternal gene expression during oocyte maturation. Key words: Cytoplasmic polyadenylation, Maternal gene, Mitogen-activated protein kinase (MAPK), M phase promoting factor (MPF) (J. Reprod. Dev. 57: [49][50][51][52][53][54][55][56] 2011) n mammals, fully grown follicular oocytes are arrested at the G2 stage, which is called the germinal vesicle (GV) stage of development. The oocytes resume meiosis in response to specific signals, usually hormones, or after being released from their follicular environment. Following germinal vesicle breakdown (GVBD), the chromatin is condensed, the metaphase I (MI) spindle is organized and oocytes extrude the first polar body upon completion of meiosis I. Subsequently, oocytes develop to metaphase of meiosis II (MII) and maintain the second meiotic arrest until fertilization [1][2][3]. Protein phosphorylation and dephosphorylation play pivotal roles in the oocyte meiotic cell cycle. Maturation/M-phase promoting factor (MPF) and mitogen-activated protein kinase (MAPK) are the principal regulatory systems that drive meiotic progression of oocytes. MAPK p42 and p44, also known as extracellular signal-regulated kinase 1 and 2 (ERK1 and ERK2), are serine/threonine kinases that can be phosphorylated and activated by MAPK kinase (MEK) [1,4]. MPF is a complex composed of a catalytic subunit, p34/cdc2, with serine/threon...

Ndc80 (called Hec1 in human), the core component of the Ndc80 complex, is involved in regulation of both kinetochore-microtubule interactions and the spindle assembly checkpoint in mitosis; however, its role in meiosis remains unclear. Here, we report Ndc80 expression, localization, and possible functions in mouse oocyte meiosis. Ndc80 mRNA levels gradually increased during meiosis. Immunofluorescent staining showed that Ndc80 was restricted to the germinal vesicle and associated with spindle microtubules from the Pro-MI to MII stages. Ndc80 was localized on microtubules and asters in the cytoplasm after taxol treatment, while Ndc80 staining was diffuse after disruption of microtubules by nocodazole treatment, confirming its microtubule localization. Disruption of Ndc80 function by either siRNA injection or antibody injection resulted in severe chromosome misalignment, spindle disruption, and precocious polar body extrusion. Our data show a unique localization pattern of Ndc80 in mouse oocytes and suggest that Ndc80 may be required for chromosome alignment and spindle organization, and may regulate spindle checkpoint activity during mouse oocyte meiosis.

Calcium is one of the most ubiquitous signaling molecules, and controls a wide variety of cellular processes. It is mainly stored in the endoplasmic reticulum (ER), bound to lumenal proteins. Calreticulin is the major Ca(2+)-binding chaperone in oocytes, and is integral to numerous cellular functions. To better understand the role of the ER- calreticulin-Ca(2+) pathway in oocyte maturation and early embryogenesis, we characterized the porcine calreticulin gene and investigated its expression profile during oocyte maturation and early embryonic development. Calreticulin was widely expressed in pig tissues and its transcripts were downregulated during maturation, especially at 44 hr, and were undetectable at the blastocyst stage. We also investigated the effect of increased cytosolic Ca(2+) induced by the Ca(2+)-ATPase inhibitor, cyclopiazonic acid (CPA), on pig oocyte maturation and maternal gene expression. CPA at 10 microM did not inhibit germinal vesicle breakdown, but did result in the arrest of 38.6% oocytes at or before the MI stage. In addition, expression of the maternal genes C-mos, BMP15, GDF9, and Cyclin B1 was significantly increased in CPA-treated MII oocytes compared with control groups. These data were supported by the results of poly(A)-test PCR, which revealed that the cyclin B1 short isoform (CB-S), GDF9, and C-mos underwent more intensive polyadenylation modification in CPA-treated oocytes than control oocytes, suggesting that polyadenylation may influence Ca(2+)-modulated changes in gene expression. Furthermore, CPA treatment decreased the percentage of four-cell parthenotes that developed into blastocysts, suggesting the need for functional SR/ER Ca(2+)-ATPase pumps or Ca(2+) signals during early embryo development after zygotic genome activation. Together, these data indicate that ER-calreticulin-associated Ca(2+) homeostasis plays a role in oocyte and embryo development, and that alterations in maternal gene expression may contribute to the underlying molecular mechanism, at least partially, via polyadenylation.