The role of WNT signaling in adult ovarian folliculogenesisWingless-type mouse mammary tumor virus integration site WNT signaling molecules are locally secreted glycoproteins that play important role in regulation of ovarian follicle maturation and steroid production. Components of the WNT signaling pathway have been demonstrated to impact reproductive functions, including embryonic development of the sex organs and regulation of follicle maturation controlling steroidogenesis wnt signaling and the regulation of ovarian steroidogenesis the postnatal ovary. Emerging evidence underscores the complexity of WNT signaling molecules in regulation of dynamic changes that occur in the ovary during the caudal steroid injection cycle. While disruption in the WNT signaling cascade has been recognized to have deleterious consequences to normal sexual development, more recent studies are beginning to highlight the importance of these molecules in adult ovarian function related to follicle wnt signaling and the regulation of ovarian steroidogenesis, corpus luteum formation, steroid production and fertility. Hormonal regulation of WNT genes and expression of members of the WNT signaling network, including WNT ligands, frizzled receptors, and downstream signaling components that are expressed in the postnatal ovary at distinct stages of the estrous cycle suggest a crucial role in normal ovarian function. This review will focus on the multiple functions of WNT signaling in folliculogenesis in the adult ovary. The adult ovary is a dynamic organ undergoing constant changes throughout the estrous cycle as follicles progress from immature preantral follicles to more developed preovulatory follicles and eventually formation of the corpus luteum following ovulation.
The role of WNT signaling in adult ovarian folliculogenesis
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.
Results support temporal and hormonal regulation and a potential role for WNT signaling in potentiating FSH action during dominant follicle selection. February 12, ; Accepted: May 22, ; Published: This is an open-access article distributed under the terms of the Creative Commons Attribution License , which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. The authors have declared that no competing interests exist. The precise mechanisms regulating the species-specific number of follicles that grow and ovulate while all other growing follicles undergo atresia are poorly understood. In single-ovulating species like cattle and humans, antral follicle growth occurs in a characteristic wave-like pattern  — .
The onset of each follicular wave is preceded by a transient increase in serum FSH that promotes emergence of a cohort of small antral follicles. In the face of declining FSH concentrations, typically, a single dominant follicle out of this cohort continues to grow to ovulatory size and produces increased amounts of estradiol.
Selection of a single dominant follicle is an evolutionarily conserved mechanism critical to control of number of offspring per pregnancy in monotocous species.
While the key role of pituitary gonadotropins in mediating the wave like pattern of follicular development is well established, the intrafollicular mechanisms and regulatory molecules that are obligatory for selection of a single dominant follicle during each follicular wave have not been fully established. Enhanced capacity of the dominant follicle to produce estradiol is critical to sustain dominant follicle growth and initiate estrous behavior, the preovulatory gonadotropin surge, resumption of meiosis and ovulation .
Recently it has been shown in rodents that estradiol production is regulated by members of the wingless-type mouse mammary tumor virus integration site WNT signaling family which modulate FSH action  , . Although WNT is essential for ovarian development  , its mechanistic role in regulation of folliculogenesis in the adult ovary, particularly in the context of follicular waves, is not well understood.
WNTs are secreted ligands that transduce their signals by binding to the frizzled FZD family of G protein-coupled receptors reviewed in . The WNTs control multiple developmental processes including cell fate specification, proliferation, differentiation and apoptosis, and WNT dysregulation has been implicated in the development of cancers, particularly colorectal cancers .
In the current studies, a preliminary RNA transcriptome sequencing study designed to further elucidate potential mechanisms controlling dominant follicle selection revealed expression of multiple WNT ligands, receptors and cognate intracellular signaling molecules in bovine follicles harvested at specific stages of a follicular wave. Based on these results, we hypothesized that WNT signaling is a key mediator of FSH action linked to dominant follicle selection in cattle. To begin to test this hypothesis, the temporal and hormonal regulation of mRNA for specific WNT and FZD molecules and associated pathway components in bovine granulosa cells were tested and the effects of a small molecule inhibitor of WNT signaling on FSH action in bovine granulosa cells were investigated.
Results support a potential role for WNT signaling in regulation of FSH action and ovarian follicular selection during follicular waves in cattle. Quantitative PCR was performed using duplicate Primer sequences can be found in Table 1.
RNA sequencing was performed by the W. RPKM values were calculated for expression of genes in follicles among different stages. Three experiments were performed using our previously described  granulosa cell culture system where cells respond to FSH with a dose-dependent increase in estradiol and display increased estradiol production with time in culture .
Treatments consisted of culture medium with DMSO diluent control group or medium containing 0. Media was changed every 2 days. The experiment was replicated 4 times using ovaries obtained on different days. For this experiment, granulosa cells were isolated and cultured as described for experiment 2.
The cells were then washed, aspirated from the wells and centrifuged at g for 5 min. Intra- and inter- assay coefficients of variation were 7. The total protein concentration for supernatants was determined using a DC protein assay kit Bio-Rad. 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.
Above results support distinct temporal regulation of WNT system members during dominant follicle selection and are suggestive of a regulatory role. Given the prominent role for FSH in regulation of follicular waves in cattle, the potential contribution of WNT signaling to FSH action on bovine granulosa cells was examined.
When cells were treated with increasing concentrations of the WNT signaling inhibitor IWR-1 in the absence of FSH, a slight increase in estradiol production was observed only at the 0. Granulosa cells were treated with 0, 0. Furthermore, an approximately 1. Above results support a prominent role for WNT signaling in potentiating specific components of FSH action in bovine granulosa cells.
To further understand the potential functional role in follicular selection, the hormonal regulation of expression of select WNT system components in bovine granulosa cells was investigated. In the present study, we identified components of the WNT system expressed in granulosa cells and potentially involved in dominant follicle selection during follicular waves in cattle. We have further established an intriguing regulatory role of WNT signaling pathway in specific aspects of FSH action on bovine granulosa cells associated with follicular growth and steroidogenesis and novel insight on the hormonal regulation of expression of WNT system components in bovine granulosa cells.
Collectively, results support additional studies of mechanism of action and functional requirement of WNT signaling for follicular selection in cattle. Diameter deviation marks the initiation of divergence in growth rate and estradiol producing capacity between the F1 or largest future dominant and F2 or second largest future subordinate growing follicles resulting in acquisition of follicle dominance .
To further elucidate potential regulators of this key developmental transition during follicular waves, RNA transcriptome sequencing was utilized to characterize granulosa cell transcriptome composition in the largest F1 and second largest F2 follicles immediately prior to predeviation; PD stage versus at onset of diameter deviation OD stage.
Granulosa cell expression of numerous WNT ligands, FZD receptors, catenins and other components of the intracellular signaling pathways was revealed. A transient increase in FSH precedes the onset of each follicular wave and dominant follicle selection occurs in the face of declining FSH concentrations  — . RNA transcriptome sequencing results suggest that WNT signaling might play an important role in regulation of follicle growth during follicular waves.
Ovarian expression and a prominent role for specific WNT pathway members in ovarian development, folliculogenesis and tumorigenesis in rodents have been established . To our knowledge, expression of WNT pathway components at specific stages of a follicular wave has not been reported previously.
Previous studies demonstrated greater abundance of CTNNB1 protein in large bovine follicles of abattoir origin with high versus low follicular fluid estradiol concentrations . Although follicles were not grouped using known criteria indicative of follicle health status  ,  , size range of follicles analyzed  suggest most had reached dominant follicle stage of follicular wave.
Collectively, present results support our hypothesis about the potential role of the canonical WNT signaling pathway in regulation of granulosa cell functions associated with dominant follicle selection and or dominance.
Pleiotropic actions of FSH are critical to growth of ovarian follicles including regulation of granulosa cell proliferation, estradiol production and mediated by regulation of key target genes . Previous studies support a prominent role for WNT signaling in regulation of ovarian steroidogenesis in rodents .
Interestingly, we observed a pronounced reduction in cell numbers and estradiol production for FSH treated granulosa cells subjected to WNT inhibitor treatment, which established its functional contribution to FSH action on bovine granulosa cells. A stimulatory effect of FSH on granulosa cell numbers using this culture system has been reported previously .
Similarly, a recent study demonstrated that knockdown of CTNNB1, a key member of canonical WNT signaling pathway, in murine granulosa cells compromised the ability of FSH to promote the mobilization of CX43 into gap junctions and consequently reduced gap junction intercellular communication . FSH-induced cell proliferation and estradiol production via granulosa cells are mediated via PKA-induced phosphorylation of CREBP, CTNNB1 and other transcription factors which induce the transcription of CYP19A1 gene encoding for the aromatase enzyme required to catalyze the conversion of testosterone to estradiol  , and for CCND2 and other cell cycle regulators which modulate granulosa cell proliferation .
Given this evidence, it is likely that activation of WNT signaling helps mediate FSH stimulated estradiol production in granulosa cells in part by regulating CYP19A1 transcription through stabilization and nuclear translocation of beta-catenin transcription factor. We also have previously reported a prominent role for CARTPT in negative regulation of FSH action in subordinate follicles associated with dominant follicle selection  , .
Results support specificity in potentiation of FSH target gene expression via WNT signaling in bovine granulosa cells. We also explored the hormonal regulation by FSH of expression of select WNT pathway members and accompanying effects of inhibition of WNT signaling in bovine granulosa cells.
In summary, expression of multiple WNT system components including WNT ligands, FZD receptors and downstream signaling molecules detected in bovine granulosa cells in the current studies support a potential role for canonical and non-canonical WNT pathways during follicular waves in cattle. Observed functional requirement of WNT signaling for multiple components of FSH action detected using a pharmacological approach, coupled with enhanced expression of CTNNB1 and DVL1 and reduced expression of AXIN2 in bovine granulosa cells observed during wave progression to the early dominance stage, supports a potential important role for WNT signaling in potentiating FSH action in the face of declining FSH concentrations characteristic of dominant follicle selection.
Conceived and designed the experiments: Click through the PLOS taxonomy to find articles in your field. 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.
June 17, Copyright: Introduction The precise mechanisms regulating the species-specific number of follicles that grow and ovulate while all other growing follicles undergo atresia are poorly understood. Granulosa Cell Culture Three experiments were performed using our previously described  granulosa cell culture system where cells respond to FSH with a dose-dependent increase in estradiol and display increased estradiol production with time in culture .
Discussion In the present study, we identified components of the WNT system expressed in granulosa cells and potentially involved in dominant follicle selection during follicular waves in cattle. Author Contributions Conceived and designed the experiments: View Article Google Scholar 2. J Dairy Sci View Article Google Scholar 3. View Article Google Scholar 4. Knobil E, Neill JD, editors.
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