Selective expression ofKrasG12Din granulosa cells of the mouse ovary causes defects in follicle development and ovulation

Abstract

Activation of the RAS family of small G-proteins is essential for follicle stimulating hormone-induced signaling events and the regulation of target genes in cultured granulosa cells. To analyze the functions of RAS protein in granulosa cells during ovarian follicular development in vivo, we generated conditional knock-in mouse models in which the granulosa cells express a constitutively active Kras G12D. The Kras G12D mutant mice were subfertile and exhibited signs of premature ovarian failure. The mutant ovaries contained numerous abnormal follicle-like structures that were devoid of mitotic and apoptotic cells and cells expressing granulosa cell-specific marker genes. Follicles that proceeded to the antral stage failed to ovulate and expressed reduced levels of ovulation-related genes. The human chorionic gonadotropin-stimulated phosphorylation of ERK1/2 was markedly reduced in mutant cells. Reduced ERK1/2 phosphorylation was due, in part, to increased expression of MKP3, an ERK1/2-specific phosphatase. By contrast, elevated levels of phospho-AKT were evident in granulosa cells of immature Kras G12D mice, even in the absence of hormone treatments, and were associated with the progressive decline of FOXO1 in the abnormal follicle-like structures. Thus, inappropriate activation of KRAS in granulosa cells blocks the granulosa cell differentiation pathway, leading to the persistence of abnormal non-mitotic, non-apoptotic cells rather than tumorigenic cells. Moreover, those follicles that reach the antral stage exhibit impaired responses to hormones, leading to ovulation failure. Transient but not sustained activation of RAS in granulosa cells is therefore crucial for directing normal follicle development and initiating the ovulation process.

The ovary: basic biology and clinical implications

“…Strikingly, KRAS is expressed at high levels in the granulosa cells of both small follicles and antral follicles (follicles at the late stages of follicular development that are characterized by the presence of a fluid-filled cavity known as the antrum adjacent to the oocyte; Figures 1 and 2), but its role remains to be determined (73). Expression in granulosa cells of a constitutively active form of KRAS that is frequently associated with various cancers, including ovarian surface epithelial (OSE) cell cancer (KRAS G12D ), does not stimulate proliferation or tumor formation (73). Rather, KRAS G12D -expressing granulosa cells cease dividing, do not undergo apoptosis, and fail to differentiate; that is, they are cell-cycle arrested.…”

“…As a consequence, the FSH program of gene expression is turned off while genes controlling matrix formation and luteinization are turned on (74). Recent studies show that the LH surge stimulates not only PKA but also PI3K/AKT and RAS signaling cascades, and that each of these is critical for ovulation (73). LH rapidly induces in granulosa cells the expression of the EGF-like factors amphiregulin (AREG), betacellulin (BTC), and epiregulin (EREG) (104) in a PKA-dependent manner.…”

Inhibition of MAPK by Prolactin Signaling through the Short Form of Its Receptor in the Ovary and Decidua

“…There are several known dual specificity MKPs that dephosphorylate and inactivate MAPK isoforms in mammalian cells (46). Among these MKPs, MKP1 and MKP3 are known to be induced rapidly in granulosa cells, are inhibited by NSC 95397, and regulate ERK1/2 activity negatively (26,47). This led us to examine whether the rapid increase in the expression levels of these phosphatases could play a role in PRL-mediated inhibition of ERK1/2; however, we found no such PRL-mediated stimulation.…”

“…Generation of mice with granulosa cell-specific deletion of ERK1 and ERK2 (25) revealed a critical role for these kinases in granulosa cell differentiation and ovulation, whereas expression of a constitutively active K-RAS mutant causes impaired follicular development and premature ovarian failure, presumably because of inappropriate activation of ERK1/2 in granulosa cells of growing follicles (26).…”

Effect of interrupted endogenous BMP/Smad signaling on growth and steroidogenesis of porcine granulosa cells

“…These require further elucidation. A widely accepted opinion is that the Ras-MAPK (mitogen-activated protein kinase) pathway (activated after LH surge) plays a major role in regulating GC steroidogenesis (Fan et al, 2008;. The TGF-β superfamily members are critical in maintaining cell growth and differentiation in the ovary (Pangas et al, 2008).…”