In contrast to infertile phenotypes, healthy oocyte and embryonic development were observed in GC-specific knockout mice, similar to wildtype mice (Figure 3C, ?,3D).3D). impact on mouse fertility. In contrast, the loss of in GCs caused subfertility and impaired CL formation due to reduced LHCGR and -Catenin protein levels, triggering GC apoptosis. genes are highly expressed during this period . When ovulated oocytes are not fertilized, the apoptotic system will eliminate CL, and many apoptotic genes (mice have sex-reversed ovaries that express genes associated with testicular development, along with oocyte depletion . Since then, a range of WNT ligands have been reported to be expressed in oocytes such as WNT2, 3, 5A, 7A or B, 10B and 11, and some expression of WNT2, WNT5A, and WNT11 also present in granulosa cells [22, 23]. Though the presence of many WNT proteins has been identified in the adult ovary of rodents, many questions remain regarding their mechanistic role in ovarian follicle development. WNTLESS (also known as GPR177, or Evi), a regulator essential for intracellular WNT trafficking, is responsible for the secretion of WNT proteins from signaling cells [24, 25]. Loss of WNTLESS function impedes all WNT signals but has no effect on other signaling pathways [24, 25]. mice die in the embryonic stage resulting from body axis establishment failure . Subsequently, many conditional knockout mice were generated to study WNTLESS function in different tissue types. For example, Carpenter et al. showed that WNTLESS is not required for brain and pancreas development using Cre to remove in WNT1 expressing cells, giving rise to mid/hindbrain and craniofacial defects; Zhu et al. found that WNTLESS controlled epithelial initiation of the fungiform placode by means of -mediated oral epithelial deletion of deletion in WNT1-expressing cells resembles the double knockout of WNT1 and WNT3A as well as -Catenin deletion . It has been suggested that WNTLESS controls epithelial initiation of the fungiform Cd200 placode through signaling via 6-Carboxyfluorescein epithelial WNT ligands . However, the complexity and overlapping expression of WNT signaling cascades have, to date, prevented researchers from elucidating their function in the ovary. To address this problem, we used conditional knockout (cKO) mice to investigate the role of total WNT proteins and WNT signaling in the ovary. Specifically, we generated cKO mice in which was explicitly disrupted in oocytes (and knockout mice are an optimal model to study the role of WNT signal pathways and proteins. RESULTS WNTLESS expression in the mouse ovary Our earlier studies suggest that WNTLESS is expressed ubiquitously in mouse tissues [30, 31]. In this study, we found that WNTLESS was highly expressed in follicles, including oocytes, granulosa cells, and primordial cells (Figure 1A). This result was confirmed by western blot, which showed the levels of WNTLESS protein in GV oocytes and granulosa cells (Figure 1B). Open in a separate window Figure 1 WNTLESS expression in the ovary. (A) The immunofluorescent staining of WNTLESS in the normal ovary. Green, WNTLESS; Blue, DNA. Scale bar, 100 m. (B) The levels of WNTLESS protein in oocytes and granulosa cells are displayed by the western blot method. 200 oocytes and 106 granulosa cells were used. Efficient and specific disruption of 6-Carboxyfluorescein gene was disrupted explicitly in oocytes using (Figure 2A) and (Figure 2B) and in granulosa cells using (Figure 2C)deletion efficiency in oocytes and granulosa cells was assessed by detecting the mRNA levels in the whole ovary, oocytes, and granulosa cells, respectively. As expected, we observed a significant reduction in mRNA levels in the entire ovary and isolated oocytes from (Figure 2D), and mice (Figure 2E). Similarly, the mRNA levels were markedly decreased in both whole ovary and isolated granulosa cells from (Figure 2F). Open in a separate window Figure 2 Targeted disruption of the gene. (ACC) The hybrid scheme used to develop knockout mice. Mice carrying a targeted allele (LoxP sites flank of the allele) were crossed with or or transgenic mice to delete selectively. The gene knockout was 6-Carboxyfluorescein confirmed by PCR genotyping. The isolated genomic DNA from mouse tails was amplified with primer pairs specific for the wildtype (+) (~100 bp) and flox alleles (~200 bp) or different Cre bands (mRNA in total ovary, oocytes, and granulosa cells extracts of three knockout mice. served as the internal control gene. The data are expressed as the mean SEM. *mice are subfertile despite apparently normal ovarian function and embryo development To study the effect of oocyte-specific and GC-specific deletion of on fertility, we conducted an animal breeding assay..