Subordinate follicles (SFs) of bovine follicular waves undergo atresia due to declining FSH concentrations; however, the signalling mechanisms have not been fully deciphered. We used an FSH-induced co-dominance model to determine the effect of FSH on signalling pathways in granulosa cells of the second-largest follicles (SF in control cows and co-dominant follicle (co-DF2) in FSH-treated cows). The SF was smaller than DF in control cows while diameters of co-DF1 and co-DF2 in FSH-treated cows were similar. The presence of cleaved CASP3 protein confirmed that granulosa cells of SFs, but not of DFs and co-DFs, were apoptotic. To determine the effect of FSH on molecular characteristics of the second-largest follicles, we generated relative variables for the second largest follicle in each cow. For this, variables of SF or co-DF2 were divided by the variables of the largest follicle DF or co-DF1 in each cow. There was higher transcript abundance of MAPK1/3 and AKT1/2/3 but lower abundance of phosphorylated MAPK3/1 in SF than co-DF2 granulosa cells. Abundance of mRNA and phosphorylated protein of STAT3 was higher in granulosa cells of control SF than FSH-treated co-DF2. SF granulosa cells had higher levels of LIFR and IL6ST transcripts, the two receptors involved in STAT3 activation. Further, lower transcript abundance of interleukin 6 receptor (IL6R), another receptor involved in STAT3 activation, indicated that STAT3 activation in SF granulosa cells could be mainly due to leukemia inhibitory factor (LIF) signalling. These results indicate that atresia due to lack of FSH is associated with activated LIF–STAT3 signalling in SF granulosa cells, as FSH treatment reversed such activation.
Gustavo Freitas Ilha, Monique T Rovani, Bernardo G Gasperin, Alfredo Quites Antoniazzi, Paulo Bayard Dias Gonçalves, Vilceu Bordignon and Raj Duggavathi
Vitor Braga Rissi, Werner Giehl Glanzner, Mariana Priotto de Macedo, Lady Katerine Serrano Mujica, Karine Campagnolo, Karina Gutierrez, Alessandra Bridi, Hernan Baldassarre, Paulo Bayard Dias Gonçalves and Vilceu Bordignon
Insufficient epigenetic reprogramming is incompatible with normal development of embryos produced by somatic cell nuclear transfer (SCNT), but treatment with histone deacetylases inhibitors (HDACi) enhances development of SCNT embryos. However, the mechanisms underpinning HDACi benefits in SCNT embryos remain largely uncharacterized. We hypothesized that, in addition to enhancing reprogramming, HDACi treatment may promote expression of genes not required for early development of SCNT embryos. To test this hypothesis, RNA synthesis was inhibited by treating bovine SCNT embryos with 5,6-dichlorobenzimidazole 1-β-D-ribofuranoside (DBR), which were concomitantly treated or not with Scriptaid (Scrip; an HDACi). Development to the blastocyst stage was significantly increased by treatment with Scrip alone (26.6%) or associated with DRB (28.6%) compared to Control (17.9%). The total number of nuclei was significantly improved only in embryos that were treated with both Scrip + DRB. Nuclear decondensation after SCNT was significantly increased by DRB treatment either alone or associated with Scrip. The relative mRNA expression, evaluated during the embryo genome activation (EGA) transition, revealed that some KDMs (KDM1A, KDM3A, KDM4C and KDM6A) and DNMT1 where prematurely expressed in Scrip-treated embryos. However, treatment with Scrip + DRB inhibited early mRNA expression of those genes, as well as several other KDMs (KDM4A, KDM4B, KDM5A, KDM5B, KDM5C and KDM7A) compared to embryos treated with Scrip alone. These findings revealed that HDACi improved development in SCNT embryos compared to Control, but altered the expression of genes involved in epigenetic regulation and did not improve embryo quality. Inhibition of RNA synthesis during HDACi treatment enhanced nuclear chromatin decondensation, modulated gene expression and improved SCNT embryo quality.