| Porcine oocytes and embryos contain high levels of endogenous lipids. Lipids were represented an important source of energy during oocyte development and in sustaining the growth of the embryo during preimplantation. The presence of perilipins remains unclear in porcine oocytes. Thus, the aim of this study was to identify the localization and expression of PLIN1and PLIN2in porcine oocytes during maturation. In addition, this experiment further studied the level of transcription factor PPAR gamma mRNA expression in porcine oocytes, and to determine the effection of PPAR gamma agonist TZD on oocyte development.First, Quantitative real-time polymerase chain reaction (qRT-PCR), immunostaining and Western blot methods were used to characterize the expression and distribution patterns of PLIN1and PLIN2in porcine oocytes. The results showed that PLIN1was not detectable in porcine oocytes. PLIN2and BODIPY493/503-detected neutral lipid droplets appeared identical distribution patterns and extensive colocalization in both GV and MⅡ porcine oocytes. PLIN2protein expression was higher in GV oocytes than that in MⅡ oocytes (p<0.05), although PLIN2mRNA expression was similar in both groups. These findings suggested that PLIN2was a major lipid droplet-associated protein in porcine oocytes. In addition, this experiment further studied the level of transcription factor PPAR gamma mRNA expression during porcine maturation, and the effection of PPAR gamma agonist TZD on PLIN2mRNA expression in MⅡ porcine oocytes. The results showed that PPAR gamma mRNA expression was higher in GV oocytes than that in MⅡ oocytes (p<0.01), and PPAR gamma agonist TZD can significantly downregulated PLIN2mRNA expression in MⅡ oocytes.Second, Fuether determined the effection of PPAR gamma agonist TZD on oocyte development and explored the related pathway. First, various concentration of TZD were supplemented in porcine in vitro maturation (IVM) medium. Compared with the control, supplementation of5μmol/L TZD to IVM medium significantly increased the proportion of oocytes with PB1at the metaphase-II (MⅡ) stage (48.8%±4.3vs.59.4%±1.7)(P<0.05). Further studied5μmol/L TZDs were supplemented in different mature stage (after22h, before22h, and the entire44h), the results showed that supplemened5μmol/L TZDs in the entire IVM44h can significantly increase the nuclear maturation of oocytes when compared to the control (62.06%±3.34vs.50.59%±1.21)(P<0.05). In addition, to determine the effects of5μmol/L TZDs on cumulus expansion and oocytes developmental competence. Compared with the control, supplementation of5μmol/L TZDs to IVM medium cannot significantly influence the diamater of COCs at IVM24h (371.64μm±12.60) and subsequent parthenogenetic embryo development in terms of cleavage rate (72.3%±0.08) and blastocyst formation rate (35.7%±0.00)(P>0.05). Finally, the maturation of nuclear was also evaluated5μmol/L TZDs combined with or without FSH and LH during IVM. This positive effect of5umol/L TZDs on oocytes development was blocked in the absence of both FSH and LH (2.57%±1.05vs.65.8%±2.78)(P＜0.05).Third, Further detect the change of cAMP level in porcine oocytes after GV COCs subjected to Cryotop vitrified and thawed during IVM. Compared to fresh group, the survival rate of vitrified oocyte (82%±0.03), the first polar rate (36%±0.01) and subsequent parthenogenetic embryo development in terms of cleavage rate (41%±0.01)and blastocyst formation rate (5%±0.01) were significantly decreased in the vitrified GV COCs after IVM44h (P＜0.05). In addition, There has a normal cAMP level in fresh group, and the peak of cAMP was appeared at6h and then significantly declined at24h and matained until44h; however, the phenomenon of cAMP accumulation were presented in vitrified oocytes, and the most cAMP level was emerged at24and sinificantly declined until44h.Above all, PLIN2is one of the major lipid droplets related proteins in porcine oocytes, and involved in a dynamic recombination of lipid droplets during porcine oocyte maturation. PPAR gamma agonist TZDs was combined with FSH and LH to effectively promote nuclear maturation. The cAMP level was abnormal in oocytes derived from vitrifief/thawed COCs, which may be compromised viability of porcine oocytes. |
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