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Seminiferous tubule degeneration and infertility in mice with sustained activation of WNT/CTNNB1 signaling in sertoli cells

ABSTRACT: To elucidate the role(s) of WNT/CTNNB1 signaling in the testis, transgenic Ctnnb1 tm1Mmt/+;Amhr2 tm3(cre)Bhr/+ mice were generated to obtain sustained activation of the WNT/CTNNB1 pathway in Sertoli cells. Male Ctnnb1 tm1Mmt/+;Amhr2 tm3(cre)Bhr/+ mice were sterile because of testicular atrophy starting at 5 wk of age, associated with degeneration of seminiferous tubules and the progressive loss of germ cells. Sustained WNT/CTNNB1 pathway activation was obtained in Ctnnb1 tm1Mmt/+;Amhr2 tm3(cre)Bhr/+ Sertoli cells. Sertoli cells often exhibited morphological characteristics suggestive of incomplete differentiation that appeared in a manner coincident with germ cell loss, accompanied by an increase in the expression of the immature Sertoli cell marker AMH. Microarray analyses performed on cultured Sertoli cells showed that CTNNB1 induces the expression of genes associated with the female sex determination pathway as well as cell-cycle associated genes . Together, these data suggest that the WNT/CTNNB1 pathway regulates Sertoli cell functions critical to their capacity to support spermatogenesis and to proliferate in the postnatal testis.

ORGANISM(S): Mus musculus

PROVIDER: GSE28402 | GEO | 2011/04/05

SECONDARY ACCESSION(S): PRJNA139177

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:Stable activation of the WNT signaling effector beta-catenin (CTNNB1(ex3) in ovarian granulosa cells results in the formation of premalignant lesions that develop into granulosa cell tumors (GCTs) spontaneously later in life. Loss of the tumor suppressor gene Pten accelerates GCT formation in the CTNNB1 strain. Conversely, expression of oncogenic KRASG12D causes the dramatic arrest of proliferation, differentiation and apoptosis in granulosa cells, and consequently, small abnormal follicle-like structures devoid of oocytes accumulate in the ovary. Because of the potent anti-proliferative effects of KRASG12D in granulosa cells, we sought to determine if KRASG12D would block precancerous lesion and tumor formation in follicles of the CTNNB1 mutant mice. Unexpectedly, transgenic Ctnnb1;Kras mutant mice developed early-onset GCTs leading to premature death in a manner similar to theCtnnb1;Pten mutant mice. Moreover, the GCTs in the Ctnnb1;Kras mutant mice exhibited increased GC proliferation, decreased apoptosis and impaired differentiation. Microarray and RT-PCR analyses revealed that ovaries from mice expressing dominant-stable CTNNB1 with either Pten loss or KRAS activation were unpredictably similar. Specifically, gene regulatory processes induced by CTNNB1 were mostly enhanced by either KRAS activation or Pten loss in remarkably similar patterns and degree. Furthermore, the concomitant activation of CTNNB1 and KRAS in Sertoli cells resulted in the development of granulosa cell tumors of the testis. RT-PCR studies showed a partial overlap in gene regulatory processes associated with tumor development in the ovary and testis. Together, these results suggest that KRAS activation and Pten loss induce GCT development from premalignant lesions via highly similar molecular mechanisms. four samples: average of two wild type samples (previously submitted as GSM403220 and GSM403221), beta-Catenin constitutively active mutant, beta-Catenin;Pten double mutant, and beta-Catenin;Kras(G12D) double mutant

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Seminiferous tubule degeneration and infertility in mice with sustained activation of WNT/CTNNB1 signaling in sertoli cells

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