Doxorubicin, a widely used chemotherapeutic agent, were found rapidly undergo morphological and biochemical changes via discrete effector signaling pathways consistent with the occurrence of apoptosis of oocyte, and a little known is actions of this drug in early embryos. Poly (ADP-ribose) polymerase (PARP), a DNA repair enzyme, also plays the important role during the apoptosis of cell. The cleavage of PARP by caspase-3 inactivates it and inhibits PARP's DNA-repairing abilities. Cleaved PARP (cPARP) can be a marker of apoptosis.Doxorubicin inhibited the early embryo development, but the treatment could still reach the BL (blastocyst) stagethat suggested that involved in DNA synthesis and repaired progress. Herein, the higher expression of PARP family shown especially in 2, 4 cell stagy. There was evidence of expression of Caspase3 and Bcl2l1 during embryogenesis (2 cell, 4 cell, morula and BL stage), suggesting that modulationsof apoptosis-related genes and PARP were cause by DXR. Furthermore, the effect of doxorubicin on early embryo development was assessed different stage rates, and apoptosis index also conformed doxorubicin modulate embryo development by regulating apoptosis- related genes and PARP family genes. In conclusion, Doxorubicin blocked pre- implantation development in early mouse embryos by altering apoptosis-related gene expression and inactivating DNA repair by Parp.

It is well established that mammalian cumulus cell (CC) expansion requires BMP15 (bone morphogenetic protein bone morphogenetic protein 15) and GDF9 (growth differentiation factor 9). However, the mechanisms of the factors in CC expansion are largely unclear. This study was conducted to examine the two paracrine factors and their receptor SMAD intracellular signaling mechanism of mediating porcine CC expansion and oocyte maturation, and to compare COCs (Cumulus–oocyte complexes) maturation to DOs (Denuded oocytes). COCs and DOs were in vitro matured in medium with FSH, LH and TGFB superfamily antagonists. Our results showed that the expansion of COCs was unaffected by addition of GDF9 and BMP15 recombinant protein, but cumulus cell proliferation and DOs maturation rate were enhanced. The mRNA expressions of SMAD receptor confirmed that oocytes secreted factors that activate SMAD3,4 and SMAD1 in granulosa cells and oocytes, but unaffected SMAD2. Treatment of COCs with a SMAD2/3 phosphorylation inhibitor (SB431542) inhibited CC expansion and expression of TNFAIP6. SB431542 also was revealed to inhibit DOs maturation. The activation of CC SMAD signaling by oocytes, and the requirement of SMAD2/3 signaling for expansion and oocyte maturation were studied in pig. Nonetheless, porcine oocyte maturation without SMAD2/3 signaling is likely to be needed for optimal matrix formation, but also BMP15 and GDF9 is likely to be needed in oocyte.

Doxorubicin, a widely used chemotherapeutic agent, were found rapidly undergo morphological and biochemical changes via discrete effector signaling pathways consistent with the occurrence of apoptosis of oocyte. In this report, we elucidated the molecular requirements for actions of this drug in early embryos. Poly (ADP-ribose) polymerase (PARP), a DNA repair enzyme, and its homologues have recently been shown in female oocyte cells. However, the cleavage of PARP by caspase-3 inactivates it and inhibits PARP's DNA-repairing abilities. Cleaved PARP (cPARP) may be considered a marker of apoptosis. Doxorubicin inhibited the early embryo development, but the treatment could still reach the BL (blastocyst) stage that suggested that involved in DNA synthesis and repaired progress. Herein, the higher expression of PARP family shown especially in 2, 4 cell stagy. There was evidence of expression of Caspase3 and Bcl2l1 during embryogenesis (2 cell, 4 cell, morula and BL stage), suggesting that modulations of apoptosis-related genes and PARP were cause by DXR. Furthermore, the effect of doxorubicin on early embryo development was assessed different stage rates, and apoptosis index also conformed doxorubicin modulate embryo development by regulating apoptosis-related genes and PARP family genes. In conclusion, Doxorubicin blocked pre-implantation development in early mouse embryos by altering apoptosis-related gene expression and inactivating DNA repair by Parp.

Superovulation, or ovarian stimulation is a commonly used ART for treatment of human infertility/subfertility. Recent studies suggest that superovulation unaffects methylated imprints acquisition in mouse oocytes during oogenesis, whereas disrupts DNA methylation maintenance in embryos during preimplantation development. However, the mechanisms of defects in methylation maintanence caused by superovulation remain largely unclear. We hypothesized that superovulation may disrupt the expression of DNA methyltransferases (Dnmts), the enzymes which catalyze DNA methylation acquisition and maintenance. The mice were subjected to superovulate with low (6 IU) and high (10 IU) dosage hormone. We examined the global DNA methylation levels in zygotes and DNA methylation of repeated sequences (IAP and Line 1) in blastocyst stage embryos. In addition, we investigated the expression of Dnmts (Dnmt3a, Dnmt3b, Dnmt3l and Dnmt1o) in ovulated oocytes and zygotes. Through staining with antibody 5mC and Di-H3K9 coupled with confocal microscopy, we found that global methylation profiles in zygotes derived from females after low or high dosage hormone treatment were not affected when compared to control counterpart. Moreover, methylation at IAP in blastocysts also was unaffected by superovulation, irrespective of hormone dosage. In contrast, methylation level at Line 1 decreased when the females were administered by high dosage hormone. Furthermore, expression of de novo DNA methyltransferase Dnmt3a, Dnmt3b, Dnmt3L, as well as maintenance Dnmt1o in MII oocytes and zygotes was not disrupted by superovulation. Given superovulation adversely affected methylation maintenance in blastocysts during preimplantation development but with normal expression of Dnmts in oocytes and zygotes, it is indicated that defects of embryonic methylation didn’t originate from abnormal expression of Dnmts.

Autophagy is an evolutionarily conserved lysosomal pathway for degrading cytoplasmic proteins, macromolecules, and organelles, in addition to recycling protein and ATP synthesis. Although programmed cell death (PCD) is very important during embryogenesis, the mechanism underlying the dynamic development during this process remains largely unknown. In order to obtain insights into autophagy and it's relation with apoptosis in early embryo development, we first evaluated LC3 gene expression levels in mouse embryos developing in vitro. qRT-PCR revealed high expression levels from 1- to 4 cell stage embryo, and then expression decreased during morula and blastocyst formation. Indirect immunocytochemistry showed protein synthesis of LC3 in these stage embryos. Introducing of autophagy inhibitor, 3-MA (2mM) significantly decreased both developmental rate (54.85±11.0%) and total cell number (n=71±8), but increased apoptosis rate (5.68± 1.9%) at the blastocyst. Real time RT-PCR confirmed reduced expression of selected autophagy related genes, including ULK1, Atg4A, B, C, D, Atg5, Atg8, Gabarap, Atg9A, B and Atg16L. Treatment of autophagy inducer, rapamycin (50 ng/㎖) increased both mRNA expression and protein synthesis of LC3 and apoptosis rate (16.11±3.42%), but decreased developmental rates (50.16±9.78) and total cell numbers (n=60±7) as compared to control developmental rate (70.74±12.9%), Total cell number (89.8±9) and apoptotic cell death (1.11±0.7%). These results suggest that autophagy is related with apoptosis in mouse embryo, which possibly give a role for early development.

Mitochondria are important regulators of both apoptosis and autophagy. One of the triggers for mitochondrial-mediated apoptosis is the production of reactive oxygen species (ROS), which include hydrogen peroxide, superoxide, hydroxyl radical, nitric oxide, and peroxynitrite. Recently, several studies have indicated that ROS may also be involved in the induction of autophagy. In the present study, we used H2O2 to induce mitochondrial stress and examined apoptotic- and autophagic-related gene expression and observed LC3 protein (autophagosome presence marker) expression in porcine parthenotes developing in vitro. In porcine four-cell parthenotes cultured for 5 days in NCSU37 medium containing 0.4% BSA, the developmental rate and mitochondrial distribution did not differ from that of the group supplemented with 100 μM H2O2 but significantly decreased in the group supplemented with 500 μM H2O2 (P<0.05). Transmission electron microscopy (TEM) indicated that whereas normal shaped mitochondria were observed in blastocysts from the control group, abnormal mitochondria (mitophagy) and autophagic vacuoles were observed in blastocysts from the group that received 500 μM H2O2. Furthermore, addition of H2O2 (100 μM and 500 μM) decreased cell numbers (P<0.05) and increased both apoptosis (P<0.05) and LC3 protein expression in the blastocysts. Real time RT-PCR showed that H2O2 significantly decreased mRNA expression of anti-apoptotic gene Bcl-xL but increased pro-apoptotic genes, Caspase 3 (Casp3) and Bak, and autophagy-related genes, microtubule-associated protein 1 light chain 3 (Map1lc3b) and lysosomal-associated membrane protein 2 (Lamp2). However, the addition of H2O2 had no effect on mRNA expression levels in nuclear DNA-encoded mitochondrial-related genes, cytochrome oxidase (Cox) 5a, Cox5b, and Cox6b1, but decreased mitochondrial DNA-encoded genes, D-loop (Dloop) and cytochrome b (Cytb), in blastocysts. These results suggest that H2O2 leads to mitochondrial dysfunction that results in apoptosis and autophagy, which is possibly related to porcine early embryo development.

Spc25 is a component of the Ndc80 complex which consists of Ndc80, Nuf2, Spc24, and Spc25. Previous work has shown that Spc25 is involved in regulation of kinetochore microtubule attachment, localization of Ndc80, and the spindle assembly checkpoint in mitosis. The role of Spc25 in meiosis remains unknown. Here, we report its expression, localization and functions in mouse oocyte meiosis. The Spc25 mRNA level gradually increased from the GV to MI stage, but decreased by MII during mouse oocyte meiotic maturation. Immunofluorescent staining showed that Spc25 was restricted to the germinal vesicle, and associated with chromosomes during all stages after GVBD. Overexpression of Spc25 resulted in oocyte meiotic arrest, chromosome misalignment and spindle disruption. Conversely, Spc25 RNAi resulted in precocious polar body extrusion and caused severe chromosome misalignment and aberrant spindle formation. Spc25 RNAi affected Ndc80 localization, but Ndc80 RNAi did not affect Spc25 localization.Survivin MO caused Ndc80 dispersion but did not affect localization of Spc25. Our data suggest that Spc25 is required for chromosome alignment, spindle formation, and spindle checkpoint activity through the regulation of Ndc80, but that Spc25 function is independent of survivin during meiosis.