| It is the main way to produce porcine embryos in vitro combining technologies such as in vitro maturation (IVM), in vitro fertilization, (IVF) and in vitro culture (IVC) and so forth. In practice, ovaries transportation, oocyte collection and manipulation as well as in vitro fertilization always need a duration which may cause temperature alteration for the cells exposured to their surroundings and unavoidably yield the cold stress responses. But the oocytes, especially for the pigs, are extremely susceptive to the effect of temperature changes, so these variations would result in low maturation quality and adverse effect on developmental competence after fertilization. Many previous studies focused on the influence of cold stress on animal oocytes, but most concentrated on the in vivo level shuch as cold stress on individual animals; and rarely on the cellular level which study its impact to in vitro culture of oocytes, the maturation rate, for example; changes of the subcellular structure as well as cold inducible RNA-binding protein (CIRP) expression after cold stress in pigs are also less repeoted. Most of gene expression and protein production would be suppressed and growth or multiplication of cells slowed down in the circumstance of cold stress. But some genes, such as cold shock proteins (CSPs), would express heavily which may have the functional regulating and cellular protecting abilities under cold stress. Cold inducible RNA-binding protein (CIRP) is one of the few cold shock proteins discovered in mammals. CIRP was first recognized in mice testis cells by Nishiyama in 1997 and has be demonstrated the function of cell protection during cold stress. Studied have also showed that CIRP presented constitutive expression and gene regulating trait in cells. Furthermore, CIRP would be overexpressed when cells suffer from cold stress. Currently, CIRP has been discovered in xenopus laevis, bullfrog, mice, rat and human being. Study of CIRP expression could contribute to the genetic regulation and mechanism research of cold stress. So it could be speculated that expression of CIRP may increase when porcine oocytes encountered cold stress, which could promote its cold stress tolerance. But the detailed process need study to elaborate.The low temperature used in the study (25℃ and 32℃) was determined according to previous papers which was usually adopted to treat the cells. This experiment aimed to study the effect of cold stress on the maturation rate, fertilization rate and developmental competence; subcellular changes such as chromosome, spindle and microfilament; CIRP expression changes of in vitro culturing oocytes in pigs by real-time PCR. The regulation and mechanism of CIRP expression was also discussed in the present paper. Content and its corresponding results of the study involve:1. Effect of cold stress on maturation and development of oocytes in pigPorcine oocytes were collected by syringe aspiration from slaughtering ovaries. The oocytes were cooled in 32℃ and 25℃ for 30 min,1 h and 2 h. Oocytes cultured in 38.5℃ were conducted as control. Suvival rate was tested by FDA staining and the rates MⅡ maturation, in vitro ferlization, cleavage and blastocyst were observed by culturing after cold stress. The results showed that no significant changes were observed in survival rate; maturation and fertilization rate after cold stress has no statistically alternation; in vitro blastocyst rate was also not declined obviously compared with control 0 except for 32℃ 2 h (9.21%) and 25℃ 2 h (9.71%).2. Effect of different holding medium on the oocyte development after cold stress in pigThis experiment aimed to compare the holding ability of TCM199 and pFF for pig oocytes druing cold stress. GV oocytes were cooled under ambient temperature (25℃) in TCM199 (without Cys, pFF and hormones) and pFF repectively for 0.5,2, 6,12 and 24 h. Changes of COCs morphology, MⅡ maturation, parthenogenetic activation cleavage and blastocyst rate were observed after cold stress. The results showed TCM199 tended to superior to pFF for the preservation of pig oocytes under cold stress. Oocytes preserved in pFF took off large quantities of granulosa cells and inclined to have light cytoplasm color; MⅡ maturation rate of oocytes in TCM199 for 6,12 and 24 h (57.3%,58.0%,55.0%) were significantly declined compare with control group (77.4%, P<0.05), and the ratio of pFF oocyts for 6,12 and 24 h got a very significant reduction (51.1%,52.9%,48.8%); no obvious decrease in cleavage rate was got among these groups except for pFF 24 h; but in blastocyst rate, and very significant decline to pFF 6,12,24 h (4.4%,0%,0%, P<0.01) compared with control (22.2%).3. Changes of cytoskeleton in pig oocytes after cold stressChromosome, spindle and microfilament configuration of pig oocytes were observed by LSCM after cooling at 32℃ or 25℃ for 0,0.5,1 and 2 h. The results demonstrated that changes of cytoskeleton were related with the time of cold stress. There is a significant decrease of normal ratio of chromosome and spindle configuration in 32℃ 2 h and 25℃ 2 h group (51.43% and 53.57%, P<0.05) but no obvious changes in other cold stress groups as compared with the control (77.17, P>0.05). Similarily, a significant decrease of normal ratio of microfilament configuration was showed in 25℃ 2 h group (76.78%, P<0.05) but no obvious changes in other cold stress groups compared with control (90.22%, P>0.05).4. Effect of cold stress on CIRP expression of pig oocytesTo study the regulation of CIRP expression in pig oocytes under cold stress, mRNA of CIRP was analyzed by real-time PCR in different cooling oocytes (cooled at 32℃ for 0,0.5,1,2,4,6,8,10 and 12 h or cooled for 2 h at 32℃ and 25℃). The results showed that mRNA expression of CIRP increased significantly when oocytes got cold stress (P<0.05). Furthermore, quantity of CIRP expression was related to the cooling time and temperature. Maxium expression was got at 32℃ for 4 h, and would decrease gradually after 4 h. Cold stress at 25℃ had a little lower expression than at 32℃, but did not show significant difference.5. Function of CIRP expression of pig oocytes during cold stressPorcine GV oocytes were cooled at 32℃ for 0,2,6h, and the changes of mRNA expression of CIRP, Bcl-2 and SOD-1 were tested to discover the basic mechanism of CIRP function during cold stress. The result demonstrated the correlation between CIRP and Bcl-2, SOD-1 expression change in pig oocytes under cold stress. Expression of Bcl-2 or SOD-1 would increase accompany with CIRP rising; simultaneously, when expression of CIRP decreased, Bcl-2 or SOD-1 expression reduced correspondingly. The result may reflect that overexpression of CIRP under cold stress is related with its anti-apoptosis and antioxidant ability. |
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