To investigate the causes of the occurrence and persistence of porcine cystic follicles, we evaluated the apoptosis and proliferation of follicular cells in these cysts. Apoptotic frequencies were examined by TUNEL assay and the expression of apoptosis regulators (XIAP, bax, bc1-2 and caspase-3) by immunohistochemistry, Western blotting and real-time quantitative PCR; cell proliferation activity was evaluated by PCNA immunohistochemistry and proliferation of in vitro cultured granulosa and theca cells. The low apoptotic frequency and weak proliferative activity were found in cystic follicles. Low frequency of apoptosis might be associated with decreased amounts of apoptotic-related factors (bax and caspase-3) and increased amounts of anti-apoptotic factors (XIAP and bcl-2) in cystic follicles. Significantly lower proliferation activity was detected in granulosa and theca cells from cystic follicles, and lesser PCNA-positive cells were found in cystic follicles. Our results indicate that the programmed cell death and cell proliferation system were altered in cystic follicles. The disorder between apoptosis and proliferation was responsible for maintaining a static condition without degeneration, which leads to the long-term persistence of follicles. These findings provide important novel insights into the pathogenesis of follicular cysts in sows.
Histone deacetylase 1 (HDAC1) is one of the most conserved enzymes present in the nuclei of cells, including bovine oocytes and pre-implantation embryos. However, the biological functions of HDAC1 in supporting the growth and development of bovine oocytes and embryos are still not fully elucidates. In this study, three siRNAs (si299, si672, and si1272) targeting to HDAC1 mRNA sequence were designed. After transfection into bovine fibroblast cells, si299, the most effective one in HDAC1 knock-down, was selected. The selected siRNA was microinjected into bovine germinal vesicle (GV) stage oocytes to determine the functions of HDAC1 in the maturation of bovine oocytes. Finally, the siRNA was microinjected into mature oocytes, which were then parthenogenetically activated and cultured in vitro until the blastocyst stage. The rates of cleavage, blastocyst development and acetylation of lysine 14 of H3 (H3K14) state were checked. The results suggest that HDAC1 knock-down in oocytes did not influence the rates of maturation or cleavage of parthenogenetic embryos. However, the rates of blastocyst decreased after siRNA microinjection. Furthermore, histone H3K14 acetylation level increased after siRNA microinjection into parthenogenetic embryos.
High-pressure in situ energy-dispersive x-ray diffraction experiments on GaN nanocrystals with 50 nm diameter have been carried out using a synchrotron xray source and a diamond-anvil cell up to about 79 GPa at room temperature. A pressure-induced first-order structural phase transition from the wurtzite-type structure to the rock-salt-type structure starts at about 48.8 GPa. The rock-salttype phase persists to the highest pressure in our experimental range.
Recently, we demonstrated that two members of neurotrophins, nerve growth factor and brain-derived neurotrophic factor, and two types of receptor, tyrosine kinase A (TrkA) and tyrosine kinase (TrkB), exist in ejaculated bull spermatozoa, and play a crucial role in the normal function of spermatozoa. Neurotrophin-4 (NT-4) is another neurotrophic factor that signals predominantly through the TrkB receptor tyrosine kinase, and no reports of detection of NT-4 in spermatozoa have been published. In the present study, the presence of NT-4 in mature bull spermatozoa was investigated using RT-PCR, immunofluorescence and Western blotting. The result shows that there was no RT-PCR evidence for NT-4 transcripts in bovine spermatozoa. However, the NT-4 protein was present in bovine spermatozoa, and the NT-4 immunoreactivity was localized to the equatorial segment and midpiece of bovine spermatozoa. In addition, effects of NT-4 on function of spermatozoa were studied. Significant increased mitochondria activity of mature bovine spermatozoa was observed in response to 300 or 500 ng/ml exogenous NT-4 (p < 0.05), in comparison with the control, while addition of inhibitors (40 ng/ml k252α) specific for tyrosine protein kinase significantly blocked the increase of mitochondria activity. However, NT-4 had no effects on the viability or acrosome reaction of spermatozoa (p > 0.05). Consequently, this study provided evidence that NT-4 protein was presented in the mature bull spermatozoa and can influence the mitochondrial activity of bovine spermatozoa through TrkB tyrosine kinase-dependent pathways.
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