No abstract

Background High-temperature requirement protease A2 (HtrA2/Omi) is a mitochondrial chaperone highly conserved from bacteria to humans. It plays an important role in mitochondrial homeostasis and apoptosis. In this study, we investigated the role of HtrA2 in mouse oocyte maturation. Methods The role of HtrA2 in mouse oocyte maturation was investigated by employing knockdown (KD) or overexpression (OE) of HtrA2 in young or old GV oocytes. We employed immunoblotting, immunostaining, fluorescent intensity quantification to test the HtrA2 knockdown on the GV oocyte maturation progression, spindle assembly checkpoint, mitochondrial distribution, spindle assembly, chromosome alignment, actin polymerization, DNA damage and chromosome numbers, the level of acetylated tubulin. Results We observed a significant reduction in HtrA2 protein levels in aging germinal vesicle (GV) oocytes. Young oocytes with low levels of HtrA2 due to siRNA knockdown were unable to complete meiosis and were partially blocked at metaphase I (MI). They also displayed significantly more BubR1 on kinetochores, indicating that the spindle assembly checkpoint was triggered at MI. Extrusion of the first polar body (Pb1) was significantly less frequent and oocytes with large polar bodies were observed when HtrA2 was depleted. In addition, HtrA2 knockdown induced meiotic spindle/chromosome disorganization, leading to aneuploidy at metaphase II (MII), possibly due to the elevated level of acetylated tubulin. Importantly, overexpression of HtrA2 partially rescued spindle/chromosome disorganization and reduced the rate of aneuploidy in aging oocytes. Conclusions Collectively, our data suggest that HtrA2 is a key regulator of oocyte maturation, and its deficiency with age appears to contribute to reproduction failure in females.

Background High-temperature requirement protease A2 (HtrA2/Omi) is a mitochondrial chaperone that is highly conserved from bacteria to humans. It plays an important role in mitochondrial homeostasis and apoptosis. In this study, we investigated the role of HtrA2 in mouse oocyte maturation. Methods The role of HtrA2 in mouse oocyte maturation was investigated by employing knockdown (KD) or overexpression (OE) of HtrA2 in young or old germinal vesicle (GV) oocytes. We employed immunoblotting, immunostaining, fluorescent intensity quantification to test the HtrA2 knockdown on the GV oocyte maturation progression, spindle assembly checkpoint, mitochondrial distribution, spindle organization, chromosome alignment, actin polymerization, DNA damage and chromosome numbers and acetylated tubulin levels. Results We observed a significant reduction in HtrA2 protein levels in aging germinal vesicle (GV) oocytes. Young oocytes with low levels of HtrA2 due to siRNA knockdown were unable to complete meiosis and were partially blocked at metaphase I (MI). They also displayed significantly more BubR1 on kinetochores, indicating that the spindle assembly checkpoint was triggered at MI. Extrusion of the first polar body (Pb1) was significantly less frequent and oocytes with large polar bodies were observed when HtrA2 was depleted. In addition, HtrA2 knockdown induced meiotic spindle/chromosome disorganization, leading to aneuploidy at metaphase II (MII), possibly due to the elevated level of acetylated tubulin. Importantly, overexpression of HtrA2 partially rescued spindle/chromosome disorganization and reduced the rate of aneuploidy in aging GV oocytes. Conclusions Collectively, our data suggest that HtrA2 is a key regulator of oocyte maturation, and its deficiency with age appears to contribute to reproduction failure in females.

Accumulating evidence has demonstrated that high concentration of benzo(a)pyrene (BaP) causes oocyte and embryo developmental arrest or death resulting in early pregnancy loss. However, whether the physiological concentration of BaP exposure affects GV oocyte maturation in exposure population remains unclear. Here, we evaluate the effects of human ovarian follicular fluid concentrations of BaP on mouse and human germinal vesicle (GV) oocyte maturation. Moreover, we examined whether nicotinic acid (NA) reversed GV meiotic failure caused by BaP during in vitro maturation (IVM). We used human ovarian follicular fluid concentrations of 5 nM BaP and/or a relatively high concentration of 50 nM group to treat GV oocytes during IVM in mice and human. We found 5 nM/50 nM BaP exposure significantly reduced first polar body extrusion during mouse GV oocytes maturation. Sirt1 protein expression decreased after BaP treatment in mouse oocytes. Moreover, BaP exposure disorganized spindle and chromosome arrangement, disrupted cortical actin cap, impaired mitochondrial redistribution, and caused DNA damage in IVM metaphase II (MII) mouse oocytes. Importantly, NA supplementation (15µM) increased Sirt1 expression and significantly rescued most of the abnormal effects. We then explored the effect of 5 nM BaP on human GV oocytes, a concentration close to that in human ovarian follicular fluid, and found that BaP caused GV meiotic failure by increasing mitochondrial membrane potential and markedly elevating reactive oxygen species (ROS) levels. Finally, we showed that 15 µM NA supplementation partially rescued human GV oocytes from the toxicity of 5 nM BaP during IVM. Our study indicates that physiological concentrations of BaP could seriously disrupt GV oocyte IVM and cause meiotic defects leading to oocyte arrest in both mice and humans. NA partially protects GV oocyte meiosis against BaP toxicity during IVM.