Restraint-induced corticotrophin-releasing hormone elevation triggers apoptosis of ovarian cells and impairs oocyte competence via activation of the Fas/FasL system.

Mechanisms by which psychological stress damages oocytes are largely undetermined. Although a previous study showed that the stress-induced corticotrophin-releasing hormone (CRH) elevation impaired oocyte competence by triggering apoptosis of ovarian cells, how CRH causes apoptosis in ovarian cells and oocytes is unknown. In this study, we have examined the hypothesis that restraint stress (RS)-induced CRH elevation triggers apoptosis of ovarian cells and impairs oocyte competence through activating the Fas/FasL system. The results showed that RS of female mice impaired oocyte competence, enhanced expression of CRH and CRH receptor (CRH-R) in the ovary, and induced apoptosis while activating the Fas/FasL system in mural granulosa cells (MGCs) and oocytes. Injecting mice with CRH-R1 antagonist antalarmin significantly alleviated the adverse effect of RS on oocyte developmental potential. Treatment of cultured MGCs recapitulated the effects of CRH and antalarmin on apoptosis and Fas/FasL expression in MGCs. Silencing FasL gene by RNA interference in cultured MGCs further confirmed the involvement of the Fas/FasL system in the CRH triggered apoptosis of ovarian cells. It is concluded that the RS-induced CRH elevation triggers apoptosis of ovarian cells and impairs oocyte competence via activation of the Fas/FasL system.

Role of autophagy in modulating post-maturation aging of mouse oocytes.

Mechanisms for post-maturation oocyte aging (PMOA) are not fully understood, and whether autophagy plays any role in PMOA is unknown. To explore the role of autophagy in PMOA, expression of autophagosomes and effects of the autophagy (macro-autophagy) activity on PMOA were observed in mouse oocytes. Oocyte activation rates and active caspase-3 levels increased continuously from 0 to 18 h of in vitro aging. While levels of microtubule-associated protein light chain 3 (LC3)-II increased up to 12 h and decreased thereafter, contents of p62 decreased from 0 to 12 h and then elevated to basal level by 18 h. However, the LC3-II/I ratio remained unchanged following aging in different media or for different times. During in vitro aging up to 12 h, upregulating autophagy with rapamycin or lithium chloride decreased activation susceptibility, cytoplasmic calcium, p62 contents, oxidative stress, caspase-3 activation and cytoplasmic fragmentation while increasing developmental competence, LC3-II contents, LC3-II/I ratio, mitochondrial membrane potential, spindle/chromosome integrity and normal cortical granule distribution. Downregulating autophagy with 3-methyladenine (3-MA) produced opposite effects on all these parameters except cytoplasmic fragmentation. After 12 h of aging culture, however, regulating autophagy with either rapamycin/lithium chloride or 3-MA had no impact on oocyte activation susceptibility. It is concluded that autophagy plays an important role in regulating PMOA. Thus, during the early stage of PMOA, autophagy increases as an adaptive response to prevent further apoptosis, but by the late stage of PMOA, the activation of more caspases blocks the autophagic process leading to severer apoptosis.

Parental life events cause behavioral difference among offspring: Adult pre-gestational restraint stress reduces anxiety across generations.

While effects of gestational, neonatal or adolescent stress on psychological alterations in progeny have been extensively studied, much less is known regarding the effects of adult pre-gestational life events on offspring behavior. Although full siblings often display behavioral differences, whether the different parental life events prior to different pregnancies contribute to these behavioral differences among siblings is worth studying. In this study, male and female adult mice were restrained for 60 days before mating with unstressed or stressed partners. F1 offspring were examined for anxiety or mated to generate F2. Both F1 females and males from restrained mothers and/or fathers showed significantly reduced anxiety and serum cortisol and increased mRNA levels of glucocorticoid receptor and brain-derived neurotrophic factor compared to control offspring from unstressed parents. Similar behavioral and molecular changes were also observed in F2 females and males. Although restraint of adolescent mice reduced anxiety in F1 of both sexes, social instability of them increased anxiety predominantly in F1 females. Thus, adult pre-gestational restraint reduced offspring's anxiety across generations; different stressors on parents may cause different phenotypes in offspring; individual behaviors can depend on adult life experiences of parents.

Transplantation of VEGF165-gene-transfected endothelial progenitor cells in the treatment of chronic venous thrombosis in rats.

BACKGROUND: The organization and recanalization of thrombi is a dynamic and complex process. The aim of this research was to study the cotherapeutic effect of stem cell transplantation and gene transfection on chronic venous thrombosis. METHODS: We constructed a recombinant adenoviral vector carrying the vascular endothelial growth factor 165 (VEGF165) gene by using the pAdEasy system, which was subsequently identified and amplified. Simultaneously, endothelial progenitor cells (EPCs) were isolated from rat bone marrow using Ficoll, cultured in EBM-2MV medium, and identified. Then, the cells were transfected with the recombinant Ad-VEGF165. The EPCs were labeled with 1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine (Dil) before transplantation. A rat model of chronic vein thrombosis was developed by partial ligation of the inferior vena cava. The rats were randomly divided into 4 groups (n = 25, each): A, Ad-VEGF165/EPC-transplantation group received 1 ml (10(6)) of Ad-VEGF165/EPCs; B, EPC-transplantation group received 1 ml (10(6)) of EPCs; C, Ad/EPC-transplantation group received 1 ml (10(6)) of Ad/EPCs; D, control group received 1 ml of the transplantation medium. The thrombi and adjacent caval walls were harvested 28 days after transplantation; real-time quantitative polymerase chain reaction was used to detect the expression level of vascular endothelial growth factor (VEGF) mRNA; and western blotting was used to measure changes in VEGF protein expression. Hematoxylin-eosin staining and immunohistochemical staining were performed to detect recanalization. Neovascularization was detected by immunohistochemical staining using the antibody for von Willebrand factor (vWF), which is a component of endothelial cells. The capillary density was quantitatively determined by counting the capillaries under a high-power microscope. RESULTS: The Ad-VEGF165 was constructed, and bone-marrow-derived EPCs were cultivated and successfully identified. We determined the optimum transfection ratio that promoted the growth of EPCs. After transfection, the EPCs secreted the VEGF protein. After transplantation, the in vivo survival of EPCs and their differentiation into endothelial cells were determined by detecting the fluorescence associated with the Dil stain. VEGF mRNA was expressed in groups A, B, C and D after transplantation, and the VEGF mRNA level in group A was significantly higher than those in groups B, C and D (P < 0.05); the VEGF mRNA levels in groups B and C were significantly higher than those in group D (P < 0.05), and there was no statistical significance between the VEGF mRNA levels in groups B and C. The recanalization capillary density in group A was significantly higher than those in groups B, C (P < 0.05) and D (P < 0.01); the recanalization capillary densities in groups B and C were significantly higher than that in group D (P < 0.05). Moreover, there was no statistical significant difference between the values for groups B and C. CONCLUSIONS: The EPCs were successfully transfected by Ad-VEGF165. A suitable transfection ratio can improve the efficiency of EPCs and the possibility of promotion of angiogenesis after transplantation. Transfected EPCs caused accelerated organization and recanalization of vein thrombi.