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WNT4 is required for ovarian follicle development and female fertility

ABSTRACT: To study the physiological role of WNT4 in the postnatal ovary, a mouse strain bearing a floxed Wnt4 allele was created and mated to the Amhr2tm3(cre)Bhr strain to target deletion of Wnt4 to granulosa cells. Wnt4flox/-;Amhr2tm3(cre)Bhr/+ mice had significantly reduced ovary weights and produced smaller litters (P<0.05). Serial follicle counting demonstrated that, while Wnt4flox/-;Amhr2tm3(cre)Bhr/+ mice were born with a normal ovarian reserve and maintained normal numbers of small follicles until puberty, they had only 25.2% of the normal number of healthy antral follicles. Some Wnt4flox/-;Amhr2tm3(cre)Bhr/+ mice had no antral follicles or corpora lutea and underwent premature follicle depletion. RTPCR analyses of Wnt4flox/-;Amhr2tm3(cre)Bhr/+ granulosa cells and cultured granulosa cells that overexpress WNT4 demonstrated that WNT4 regulates the expression of Star, Cyp11a1 and Cyp19, steroidogenic genes previously identified as downstream targets of the WNT signaling effector CTNNB1. WNT4- and CTNNB1-overexpressing cultured granulosa cells were analyzed by microarray for alterations in gene expression, which showed that WNT4 also regulates a series of genes involved in late follicle development and the cellular stress response via the WNT/CTNNB1 signaling pathway. Together, these data indicate that WNT4 is required for normal antral follicle development, and may act by regulating granulosa cell functions including steroidogenesis.

ORGANISM(S): Mus musculus

PROVIDER: GSE18704 | GEO | 2009/10/23

SECONDARY ACCESSION(S): PRJNA121495

REPOSITORIES: GEO

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Project description:To study the physiological role of WNT4 in the postnatal ovary, a mouse strain bearing a floxed Wnt4 allele was created and mated to the Amhr2tm3(cre)Bhr strain to target deletion of Wnt4 to granulosa cells. Wnt4flox/-;Amhr2tm3(cre)Bhr/+ mice had significantly reduced ovary weights and produced smaller litters (P<0.05). Serial follicle counting demonstrated that, while Wnt4flox/-;Amhr2tm3(cre)Bhr/+ mice were born with a normal ovarian reserve and maintained normal numbers of small follicles until puberty, they had only 25.2% of the normal number of healthy antral follicles. Some Wnt4flox/-;Amhr2tm3(cre)Bhr/+ mice had no antral follicles or corpora lutea and underwent premature follicle depletion. RTPCR analyses of Wnt4flox/-;Amhr2tm3(cre)Bhr/+ granulosa cells and cultured granulosa cells that overexpress WNT4 demonstrated that WNT4 regulates the expression of Star, Cyp11a1 and Cyp19, steroidogenic genes previously identified as downstream targets of the WNT signaling effector CTNNB1. WNT4- and CTNNB1-overexpressing cultured granulosa cells were analyzed by microarray for alterations in gene expression, which showed that WNT4 also regulates a series of genes involved in late follicle development and the cellular stress response via the WNT/CTNNB1 signaling pathway. Together, these data indicate that WNT4 is required for normal antral follicle development, and may act by regulating granulosa cell functions including steroidogenesis. Experiment Overall Design: Granulosa cells were obtained from 20-26 day-old mice of various genotypes 48h after eCG treatment, using the needle puncture method as previously described. Cells were then infected with adenoviruses to express eGFP or Cre, or overexpress WNT4 in serum-free medium, and subsequently harvested for RNA extraction as described below. Preliminary experiments demonstrated that an infection efficiency of >80% could be obtained at an MOI of ~50 (as determined by analysis of fluorescent signal in Ad-eGFP-infected cells) and that the Ad-Cre and Ad-WNT4 viruses produced efficient recombination of the floxed Ctnnb1 alleles and robust WNT4 overexpression, respectively. Microarray analyses were done using triplicate RNA samples from each adenoviral treatment described above, and using mouse expression set 430 microarrays (Affymetrix, Santa Clara, CA).

Project description:Antral follicle size might be a valuable additive predictive marker for IVF outcome. However, while some studies show positive relations between follicle size and reproductive outcome, others have not been successful in establishing this relation. To better understand consequences of antral follicle size for reproductive outcome, we aimed to obtain insight in follicle size-related granulosa cell processes, as granulosa cells play an essential role in follicular development via the production of growth factors, steroids and metabolic intermediates, needed for follicular growth and oocyte development. Using pigs as a model, we compared gene and protein expression in granulosa cells of smaller and larger follicles in the healthy antral follicle pool at the start of the follicular phase of the estrous cycle. In sows, the early antral follicle pool is very heterogeneous when e.g. size and steroid content of the follicular fluid are considered. To which extent this variety contributes to the developmental competence of the follicles is not clear. Therefore, sows with high variation in antral follicle size (HighVAR) as well as sows with low variation in antral follicle size (LowVAR) were used. Granulosa cells of smaller antral follicles in the healthy antral follicle pool show increased cell proliferation, which was accompanied by a metabolic shift towards aerobic glycolysis (i.e. the Warburg effect), similar to other highly proliferating cells. High granulosa cell proliferation rates in smaller follicles might be regulated via increased granulosa cell expression of AR and EGFR which are activated in response to locally produced mitogens. While granulosa cells of smaller follicles in the pool were more proliferative, indicative of higher follicular growth, granulosa cells of larger follicles in the pool showed less proliferation and were more differentiated, as they showed a higher expression of follicular maturation marker IGF1 and ANGPT1. Our results imply that the inclusion of strict criteria of antral follicle size in IVF protocols might improve reproductive outcome. In addition, we have granulosa cell gene expression of healthy follicles to unravel underlying mechanisms of differences in COC morphology. We compared gene expression in sows with low vs. high-COC-health and found a decreased expression of genes involved in ovarian steroidogenesis (e.g. CYP19A1, ADM, SPP1) and higher expression of genes involved in follicular atresia (e.g. GADD45A, INHBB) in sows with low-COC-health. Thereby we have identified several genes which may serve as markers for follicle developmental competence.

Project description:Dibutyl phthalate (DBP), di-2-ethylhexyl phthalate (DEHP), and benzyl butyl phthalate (BBP) are three phthalates commonly found in consumer products, including the plastic coating of pharmaceuticals and personal care products. Folliculogenesis, a tightly regulated process occurring in the ovary, is the maturation of an immature primordial follicle to a mature antral follicle. Follicles house the oocyte and antral follicles specifically play a crucial role in ovarian steroidogenesis and ovulation. DBP, BBP, and DEHP have been associated with inhibited antral follicle growth in vitro, decreased ovulation rates in vitro, and decreased antral follicle counts in women. However, little is known about the effects of a three-phthalate mixture on antral follicles in vivo. The objective of this study was to evaluate the effects of a human relevant mixture of DBP, BBP, and DEHP on ovarian follicles through proteome profiling analysis. CD-1 female mice (60 days old) were pipet fed tocopherol stripped corn oil (vehicle control) only or a phthalate mixture (52% DBP, 36% DEHP, and 12% BBP dissolved in vehicle) which modeled human follicular fluid concentrations. The mice were treated with 32µg/kg/day (PHT Mix 32; cumulative estimate in general population) and 500µg/kg/day (PHT Mix 500; cumulative estimate in occupationally exposed individuals) for 10 consecutive days. Proteome profiling of antral follicles (>250µm) was performed via label-free tandem mass spectrometry. A total of 5,417 antral follicle proteins were identified in the three groups, of which 194 were differentially abundant between the vehicle and PHT Mix 32 group, and 136 between the vehicle and PHT Mix 500 group. Gene ontology analysis revealed that the two treatments of the phthalate mixture upregulate and downregulate distinctive processes, supporting non-monotonic effects of phthalates on the antral follicle proteome. Taken together, these results reveal that a human relevant mixture of DBP, BBP, and DEHP alters the antral follicle proteome and merits further evaluation to elucidate the molecular mechanisms by which phthalates cause negative reproductive outcomes.

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WNT4 is required for ovarian follicle development and female fertility

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