MicroRNA expression and function during porcine oocyte maturation and early embryonic development
Understanding oocyte maturation and embryo development in the pig is essential to improving farm animal reproduction efficiency. Many reproductive tissues are associated with a high degree of transcriptional and translational turnover that may be in part regulated by non-coding RNA such as microRNA (miRNA). The oocyte consists of a storage of maternal RNA and nutrients that are utilized to support oocyte maturation and early embryo development. MiRNA have been identified in the oocyte and throughout early embryo development in many species including the pig. MiRNA exert their biological impact on the cell through post transcriptional gene regulation (PTGR) by interacting with the 3' UTR of a specific target mRNA. MiRNA expression during oocyte maturation and embryo development up to the 4-cell stage is potentially critical as little to no transcription occurs in the oocyte following germinal vesicle breakdown until the activation of the embryonic genome. Our objectives were to 1) determine the importance of MIR21 expression and function during oocyte maturation, 2) develop and test an in vitro heat stress (HS) model for pig oocyte maturation and test the effects of HS on MIR21 expression, and 3) characterize the expression of miRNA during rapid trophoblastic elongation in the pig. Our data demonstrate that expression of mature MIR21 is increased approximately 4-fold (P = 0.001) in MII oocytes compared to GV stage oocytes. Additionally when MIR21 is inhibited during in vitro maturation, maturation rates are decreased from 55.4 ± 3.6% in control to 33.7 ± 3.6% in MIR21 inhibited. Following in vitro fertilization, MIR21 inhibited oocytes produced fewer 4-cell stage embryos (41.7 ± 12.1%) compared to the control group (73.0 ± 5.7%). While no decrease (P = 0.34) in PDCD4 mRNA (a MIR21 target) was observed, inhibition of MIR21 resulted in increased (P < 0.05) PDCD4 protein expression in MII oocytes compared to control MII arrested oocytes. Heat stress during oocyte maturation caused alterations in both MIR21 and PDCD4 expression in MII oocytes and in 4-cell embryos created from oocytes matured during HS. In addition, other markers of HS, such as heat shock protein 90A (HSP90A), were also affected in 4-cell stage embryos created from HS oocytes. Our data demonstrated that HS during oocyte maturation caused a significant decrease in MIR21 in 4-cell stage embryos cultured in thermal neutral conditions which was associated with a significant increase in PDCD4 mRNA in the same group of embryos compared to 4-cell stage embryos created from oocytes matured in TN conditions.
Elongating pig conceptus were collected from pregnant pigs on Day 12 of gestation (spherical and elongated) and on Day 14 (elongated; n =4 for each morphological stage). Small RNA libraries were created and subjected to massively parallel deep sequencing using the ABI SoLID sequencing platform. Total miRNA reads for each stage of development were 7,319K, 13,831K, and 30,618K for spherical (D12S), day 12 filamentous (D12F) and day 14 filamentous (D14F), respectively. Several miRNA were selected for validation including MIR21, MIR301a, MIR23b, MIR10a, MIR200a, MIR574, MIR4057, and MIR467a. Many of these selected miRNA demonstrated significant fold changes between two or more conceptus stages and were validated within individual samples at each stage by RT-PCR. This confirmed that miRNA are temporally regulated during embryo elongation and potentially play a role in regulating cell differentiation, migration and transformation of the porcine conceptus.