Ferritin heavy chain participated in ameliorating 3-nitropropionic acid-induced oxidative stress and apoptosis of goose follicular granulosa cells.

Oxidative stress is the major culprits responsible for ovarian dysfunction by damaging granulosa cells (GCs). Ferritin heavy chain (FHC) may participate in the regulation of ovarian function by mediating GCs apoptosis. However, the specific regulatory function of FHC in follicular GCs remains unclear. Here, 3-nitropropionic acid (3-NPA) was utilized to establish an oxidative stress model of follicular GCs of Sichuan white geese. To explore the regulatory effects of FHC on oxidative stress and apoptosis of primary GCs in geese by interfering or overexpressing FHC gene. After transfection of siRNA-FHC to GCs for 60 h, the expressions of FHC gene and protein decreased significantly (P < 0.05). After FHC overexpression for 72 h, the expressions of FHC mRNA and protein upregulated considerably (P < 0.05). The activity of GCs was impaired after interfering with FHC and 3-NPA coincubated (P < 0.05). When overexpression of FHC combined with 3-NPA treatment, the activity of GCs was remarkably enhanced (P < 0.05). After interference FHC and 3-NPA treatment, NF-κB and NRF2 gene expression decreased (P < 0.05), the intracellular reactive oxygen species (ROS) level increased greatly (P < 0.05), BCL-2 expression reduced, BAX/BCL-2 ratio intensified (P < 0.05), the mitochondrial membrane potential decreased notably (P < 0.05), and the apoptosis rate of GCs aggravated (P < 0.05). While overexpression of FHC combined with 3-NPA treatment could promote BCL-2 protein expression and reduce BAX/BCL-2 ratio, indicating that FHC regulated the mitochondrial membrane potential and apoptosis of GCs by mediating the expression of BCL-2. Taken together, our research manifested that FHC alleviated the inhibitory effect of 3-NPA on the activity of GCs. FHC knockdown could suppress the expression of NRF2 and NF-κB genes, reduce BCL-2 expression and augment BAX/BCL-2 ratio, contributing to the accumulation of ROS and jeopardizing mitochondrial membrane potential, as well as exacerbating GCs apoptosis.

Effects of ODC on polyamine metabolism, hormone levels, cell proliferation and apoptosis in goose ovarian granulosa cells.

Ornithine decarboxylase (ODC) plays an indispensable role in the process of polyamine biosynthesis. Polyamines are a pivotal part of living cells and have diverse roles in the regulation of cell proliferation and apoptosis, aging and reproduction. However, to date, there have been no reports about ODC regulating follicular development in goose ovaries. Here, we constructed ODC siRNA and overexpression plasmids and transfected them into goose primary granulosa cells (GCs) to elucidate the effects of ODC interference and overexpression on the polyamine metabolism, hormone levels, cell apoptosis and proliferation of granulosa cells. After interfering with ODC in GCs, the mRNA and protein levels of ODC and the content of putrescine were greatly decreased (P < 0.05). When ODC was overexpressed, ODC mRNA and protein levels and putrescine content were greatly increased (P < 0.05). The polyamine-metabolizing enzyme genes ornithine decarboxylase antizyme 1 (OAZ1) and spermidine / spermine-N1-acetyltransferase (SSAT) were significantly increased, and spermidine synthase (SPDS) was significantly decreased when ODC was downregulated (P < 0.05). OAZ1, SPDS and SSAT were significantly increased when ODC was upregulated (P < 0.05). In addition, after interference with ODC, progesterone (P4) levels in the culture medium of GCs increased greatly (P < 0.05), while the overexpression of ODC caused the P4 level to decrease significantly (P < 0.05). After ODC downregulation, granulosa cell activity was significantly reduced, the apoptosis rate was significantly increased, and the BCL-2 / BAX ratio was downregulated (P < 0.05). Under ODC overexpression, the activity of GCs was notably increased, the apoptosis rate was significantly reduced, and the BCL-2 / BAX protein ratio was upregulated (P < 0.05). Our study successfully induced ODC interference and overexpression in goose ovarian GCs, and ODC regulated mainly putrescine content in GCs with a slight influence on spermidine and spermine. Moreover, ODC participated in the adjustment of P4 levels in the culture medium of GCs, promoted granulosa cell proliferation and inhibited granulosa cell apoptosis.

Exogenous spermidine affects polyamine metabolism in the mouse hypothalamus.

Spermidine is important for the hypothalamic control of pituitary secretion of hormones involved in neuroendocrine functions in mammals. In this study, the effect of exogenous spermidine on the expression of genes and proteins related to polyamine metabolism and polyamine levels was examined. The results indicated that treatment with spermidine at 0.05 mg/g (BW) significantly increased the levels of Oaz1 mRNA and protein expression and decreased putrescine content in mouse hypothalamus (p < 0.05). The administration with spermidine at 0.10 mg/g significantly increased the levels of Oaz1, Oaz2, and Odc expression in mouse hypothalamus (p < 0.05). Treatment with spermidine at 0.05 mg/g significantly increased the levels of Ssat mRNA expression and reduced the level of Smo mRNA expression in mouse hypothalamus (p < 0.05). Putrescine concentrations in the hypothalamus after the administration of spermidine at 0.10 and 0.15 mg/g were significantly higher than those in the control group (p < 0.05). The concentration of both spermidine and spermine in the hypothalamus after the administration of spermidine at 0.15 mg/g was decreased significantly (p < 0.05). In summary, our results indicate that exogenous spermidine affects polyamine homeostasis in the mouse hypothalamus by modulating the expression of genes and proteins related to polyamine metabolism.

Spermidine at supraphysiological doses induces oxidative stress and granulosa cell apoptosis in mouse ovaries.

Given that spermidine is associated with aging-related diseases and it is a potential target for delaying aging, functional studies on supraphysiological levels of spermidine are required. Our previous studies showed that the granulosa layer arranged irregular and the follicular oocytes were shrunk in female mice injected intraperitoneally with spermidine at 150 mg/kg (Body weight) after 24 h. It indicated that supraphysiological levels of spermidine induced ovarian damage in female mice. The objective of this study was to investigate the effect of acute administration of supraphysiological spermidine on the ovary and granulosa cells in mice. The results showed that treatment with spermidine at 150 mg/kg (intraperitoneal) significantly increased the levels of both H2O2 and malondialdehyde and reduced total antioxidant capacity and the activities of catalase and superoxide dismutase in mouse ovaries. The contents of putrescine and spermine increased significantly in the ovaries of mice treated with spermidine. Treatment with spermidine at 150 mg/kg increased the apoptotic rate and reactive oxygen species levels of granulosa cells in mouse ovaries. Furthermore, the protein expression of P53, CASPASE 8 (Cleaved/Pro), CASPASE 9 (Cleaved/Pro) and CASPASE 3 (Cleaved/Pro) in granulosa cells of mice treated with spermidine were significantly upregulated, while BCL2 expression was significantly downregulated. In summary, our study demonstrates for the first time that spermidine at supraphysiological doses causes ovarian oxidative stress and induces granulosa cell apoptosis via the P53 and/or BCL2-CASPASEs pathway.

A novel thymidine phosphorylase mutation in a Chinese MNGIE patient.

Mitochondrial neurogastrointestinal encephalomyopathy (MNGIE) is an autosomal recessive disorder associated with mitochondrial alterations. MNGIE is characterized by severe gastrointestinal dysmotility, cachexia, ophthalmoplegia, ptosis, peripheral neuropathy, and leukoencephalopathy. The condition is caused by mutation of the TYMP gene. We studied the clinical and biochemical characteristics of a family with MNGIE. The proband was a 48-year-old male presenting with diarrhea and progressive weight loss. He also had ptosis and exhibited eyeball fixation. His blood and cerebrospinal fluid lactate levels were elevated. Magnetic resonance imaging of the brain revealed diffuse leukoencephalopathy. Ragged red fibers and cytochrome c oxidase-deficient fibers were apparent on muscle biopsy. His vision and ptosis deteriorated significantly during follow-up. Our clinical diagnosis of MNGIE was confirmed by TYMP gene analysis. We discovered a homozygous TYMP c.1193-1216 dup-GGGCGCTGCCGCTGGCGCTGGTGC mutation (a duplication). Some of the family members were heterozygous for the mutation but had no clinical features. We predicted the function of this mutation using PredictProtein and found that the secondary structure had changed in the region of the helix and strand, the transmembrane region, and the protein-protein binding sites. The family described herein exhibited biochemically, genetically, and functionally confirmed MNGIE syndrome.