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Regulation and regulatory role of WNT signaling in potentiating FSH action during bovine dominant follicle selection.

Gupta PS, Folger JK, Rajput SK, Lv L, Yao J, Ireland JJ, Smith GW - PLoS ONE (2014)

Bottom Line: FSH treatment increased CYP19A1, CCND2, CTNNB1, AXIN2 and FZD6 mRNAs and the stimulatory effect on CYP19A1 mRNA was reduced by IWR-1.In contrast, FSH reduced CARTPT mRNA and IWR-1 partially reversed the inhibitory effect of FSH.Results support temporal and hormonal regulation and a potential role for WNT signaling in potentiating FSH action during dominant follicle selection.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Mammalian Reproductive Biology and Genomics, Michigan State University, East Lansing, Michigan, United States of America; Department of Animal Science, Michigan State University, East Lansing, Michigan, United States of America.

ABSTRACT
Follicular development occurs in wave like patterns in monotocous species such as cattle and humans and is regulated by a complex interaction of gonadotropins with local intrafollicular regulatory molecules. To further elucidate potential mechanisms controlling dominant follicle selection, granulosa cell RNA harvested from F1 (largest) and F2 (second largest) follicles isolated at predeviation (PD) and onset of diameter deviation (OD) stages of the first follicular wave was subjected to preliminary RNA transcriptome analysis. Expression of numerous WNT system components was observed. Hence experiments were performed to test the hypothesis that WNT signaling modulates FSH action on granulosa cells during follicular waves. Abundance of mRNA for WNT pathway members was evaluated in granulosa cells harvested from follicles at emergence (EM), PD, OD and early dominance (ED) stages of the first follicular wave. In F1 follicles, abundance of CTNNB1 and DVL1 mRNAs was higher and AXIN2 mRNA was lower at ED versus EM stages and DVL1 and FZD6 mRNAs were higher and AXIN2 mRNA was lower in F1 versus F2 follicle at the ED stage. Bovine granulosa cells were treated in vitro with increasing doses of the WNT inhibitor IWR-1+/- maximal stimulatory dose of FSH. IWR-1 treatment blocked the FSH-induced increase in granulosa cell numbers and reduced the FSH-induced increase in estradiol. Granulosa cells were also cultured in the presence or absence of FSH +/- IWR-1 and hormonal regulation of mRNA for WNT pathway members and known FSH targets determined. FSH treatment increased CYP19A1, CCND2, CTNNB1, AXIN2 and FZD6 mRNAs and the stimulatory effect on CYP19A1 mRNA was reduced by IWR-1. In contrast, FSH reduced CARTPT mRNA and IWR-1 partially reversed the inhibitory effect of FSH. Results support temporal and hormonal regulation and a potential role for WNT signaling in potentiating FSH action during dominant follicle selection.

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Temporal expression profile of select WNT system components in granulosa cells harvested from F1 and F2 follicles at the emergence (EM; n = 3 animals), predeviation (PD; n = 5 animals), onset of deviation (OD; n = 5 animals) and early dominance (ED; n = 5 animals) stages of the first follicular wave Relative mRNA abundance for FZD6 (A), DVL1 (B), AXIN2 (C) and CTNNB1 (D) was determined by Q-RTPCR.RNA expression data was normalized relative to ACTIN as internal control. A,B P<0.05 for transcript abundance in F1 follicles across stages of follicular wave; X,Y P<0.05 for transcript abundance in F2 follicles across stages of follicular wave; *P<0.05 for transcript abundance between F1 versus F2 follicles within stage of follicular wave.
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pone-0100201-g001: Temporal expression profile of select WNT system components in granulosa cells harvested from F1 and F2 follicles at the emergence (EM; n = 3 animals), predeviation (PD; n = 5 animals), onset of deviation (OD; n = 5 animals) and early dominance (ED; n = 5 animals) stages of the first follicular wave Relative mRNA abundance for FZD6 (A), DVL1 (B), AXIN2 (C) and CTNNB1 (D) was determined by Q-RTPCR.RNA expression data was normalized relative to ACTIN as internal control. A,B P<0.05 for transcript abundance in F1 follicles across stages of follicular wave; X,Y P<0.05 for transcript abundance in F2 follicles across stages of follicular wave; *P<0.05 for transcript abundance between F1 versus F2 follicles within stage of follicular wave.

Mentions: Preliminary RNA sequencing based comparative transcriptome analysis of bovine follicles at predeviation (PD) and onset deviation (OD) stages has demonstrated expression of numerous WNT signaling pathways members including WNT ligand molecules, frizzled receptors and catenins (Table S1). Based on this observation, temporal expression of select members (CTNNB1, AXIN2, DVL1 and FZD6) of interest of the WNT system was further examined by real-time PCR using total RNA isolated from largest (F1) and second largest (F2) follicles collected at four different stages of a follicular wave. In the F1 follicles, relative abundance of FZD6 mRNA was ∼2 fold greater (P<0.05) in follicles collected at ED stage relative to OD stage (Figure 1A). Abundance of mRNAs for DVL1 (Figure 1B) and CTNNB1 (Figure 1D) was significantly greater (P<0.05) and that of AXIN2 (Figure 1C) was ∼2 fold lower (P<0.05) during the ED stage compared to EM stage. Real time PCR analysis of F2 follicles revealed similar expression pattern for DVL1, AXIN2 and CTNNB1 (Figure 1B-D) transcripts as observed in F1 follicles, however FZD6 transcript showed no significant variation in expression among different stages of follicular development in F2 (Figure 1A). Furthermore, mRNA abundance for FZD6 and DVL1 was higher (P<0.05) and AXIN2 was lower (P<0.05) in the F1 (dominant) versus F2 (subordinate) follicles at the early dominance stage (immediately after dominant follicle selection; Figure 1A and 1C).


Regulation and regulatory role of WNT signaling in potentiating FSH action during bovine dominant follicle selection.

Gupta PS, Folger JK, Rajput SK, Lv L, Yao J, Ireland JJ, Smith GW - PLoS ONE (2014)

Temporal expression profile of select WNT system components in granulosa cells harvested from F1 and F2 follicles at the emergence (EM; n = 3 animals), predeviation (PD; n = 5 animals), onset of deviation (OD; n = 5 animals) and early dominance (ED; n = 5 animals) stages of the first follicular wave Relative mRNA abundance for FZD6 (A), DVL1 (B), AXIN2 (C) and CTNNB1 (D) was determined by Q-RTPCR.RNA expression data was normalized relative to ACTIN as internal control. A,B P<0.05 for transcript abundance in F1 follicles across stages of follicular wave; X,Y P<0.05 for transcript abundance in F2 follicles across stages of follicular wave; *P<0.05 for transcript abundance between F1 versus F2 follicles within stage of follicular wave.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4061082&req=5

pone-0100201-g001: Temporal expression profile of select WNT system components in granulosa cells harvested from F1 and F2 follicles at the emergence (EM; n = 3 animals), predeviation (PD; n = 5 animals), onset of deviation (OD; n = 5 animals) and early dominance (ED; n = 5 animals) stages of the first follicular wave Relative mRNA abundance for FZD6 (A), DVL1 (B), AXIN2 (C) and CTNNB1 (D) was determined by Q-RTPCR.RNA expression data was normalized relative to ACTIN as internal control. A,B P<0.05 for transcript abundance in F1 follicles across stages of follicular wave; X,Y P<0.05 for transcript abundance in F2 follicles across stages of follicular wave; *P<0.05 for transcript abundance between F1 versus F2 follicles within stage of follicular wave.
Mentions: Preliminary RNA sequencing based comparative transcriptome analysis of bovine follicles at predeviation (PD) and onset deviation (OD) stages has demonstrated expression of numerous WNT signaling pathways members including WNT ligand molecules, frizzled receptors and catenins (Table S1). Based on this observation, temporal expression of select members (CTNNB1, AXIN2, DVL1 and FZD6) of interest of the WNT system was further examined by real-time PCR using total RNA isolated from largest (F1) and second largest (F2) follicles collected at four different stages of a follicular wave. In the F1 follicles, relative abundance of FZD6 mRNA was ∼2 fold greater (P<0.05) in follicles collected at ED stage relative to OD stage (Figure 1A). Abundance of mRNAs for DVL1 (Figure 1B) and CTNNB1 (Figure 1D) was significantly greater (P<0.05) and that of AXIN2 (Figure 1C) was ∼2 fold lower (P<0.05) during the ED stage compared to EM stage. Real time PCR analysis of F2 follicles revealed similar expression pattern for DVL1, AXIN2 and CTNNB1 (Figure 1B-D) transcripts as observed in F1 follicles, however FZD6 transcript showed no significant variation in expression among different stages of follicular development in F2 (Figure 1A). Furthermore, mRNA abundance for FZD6 and DVL1 was higher (P<0.05) and AXIN2 was lower (P<0.05) in the F1 (dominant) versus F2 (subordinate) follicles at the early dominance stage (immediately after dominant follicle selection; Figure 1A and 1C).

Bottom Line: FSH treatment increased CYP19A1, CCND2, CTNNB1, AXIN2 and FZD6 mRNAs and the stimulatory effect on CYP19A1 mRNA was reduced by IWR-1.In contrast, FSH reduced CARTPT mRNA and IWR-1 partially reversed the inhibitory effect of FSH.Results support temporal and hormonal regulation and a potential role for WNT signaling in potentiating FSH action during dominant follicle selection.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Mammalian Reproductive Biology and Genomics, Michigan State University, East Lansing, Michigan, United States of America; Department of Animal Science, Michigan State University, East Lansing, Michigan, United States of America.

ABSTRACT
Follicular development occurs in wave like patterns in monotocous species such as cattle and humans and is regulated by a complex interaction of gonadotropins with local intrafollicular regulatory molecules. To further elucidate potential mechanisms controlling dominant follicle selection, granulosa cell RNA harvested from F1 (largest) and F2 (second largest) follicles isolated at predeviation (PD) and onset of diameter deviation (OD) stages of the first follicular wave was subjected to preliminary RNA transcriptome analysis. Expression of numerous WNT system components was observed. Hence experiments were performed to test the hypothesis that WNT signaling modulates FSH action on granulosa cells during follicular waves. Abundance of mRNA for WNT pathway members was evaluated in granulosa cells harvested from follicles at emergence (EM), PD, OD and early dominance (ED) stages of the first follicular wave. In F1 follicles, abundance of CTNNB1 and DVL1 mRNAs was higher and AXIN2 mRNA was lower at ED versus EM stages and DVL1 and FZD6 mRNAs were higher and AXIN2 mRNA was lower in F1 versus F2 follicle at the ED stage. Bovine granulosa cells were treated in vitro with increasing doses of the WNT inhibitor IWR-1+/- maximal stimulatory dose of FSH. IWR-1 treatment blocked the FSH-induced increase in granulosa cell numbers and reduced the FSH-induced increase in estradiol. Granulosa cells were also cultured in the presence or absence of FSH +/- IWR-1 and hormonal regulation of mRNA for WNT pathway members and known FSH targets determined. FSH treatment increased CYP19A1, CCND2, CTNNB1, AXIN2 and FZD6 mRNAs and the stimulatory effect on CYP19A1 mRNA was reduced by IWR-1. In contrast, FSH reduced CARTPT mRNA and IWR-1 partially reversed the inhibitory effect of FSH. Results support temporal and hormonal regulation and a potential role for WNT signaling in potentiating FSH action during dominant follicle selection.

Show MeSH
Related in: MedlinePlus