Paxillin knockout in mouse granulosa cells increases fecundity

Abstract

Paxillin is an intracellular adaptor protein involved in focal adhesions, cell response to stress, steroid signaling and apoptosis in reproductive tissues. To investigate the role of paxillin in granulosa cells, we created a granulosa-specific paxillin knockout mouse model (GC-PXN KO) using Cre recombinase driven by the Anti-Müllerian hormone receptor 2 gene promoter. Female GC-PXN KO mice demonstrated increased fertility in later reproductive age, resulting in higher number of offspring when bred continuously up to 26 weeks of age. This was not due to increased numbers of estrous cycles, ovulated oocytes per cycle, or pups per litter, but due to shorter time to pregnancy and increased number of litters in the GC-PXN KO mice. The number of ovarian follicles was not significantly affected by the knockout at 30 weeks of age. GC-PXN KO mice had slightly altered estrous cycles but no difference in circulating reproductive hormone levels. Knockout of paxillin using CRISPR-Cas9 in human granulosa-derived immortalized KGN cells did not affect cell proliferation or migration. However, in cultured primary mouse granulosa cells, paxillin knockout reduced cell death under basal culture conditions. We conclude that paxillin knockout in granulosa cells increases female fecundity in older reproductive age mice, possibly by reducing granulosa cell death. This study implicates paxillin and its signaling network as potential granulosa cell targets in the management of age-related subfertility.

Paxillin regulates androgen receptor expression associated with granulosa cell focal adhesions

“…S3 ). We also measured AR expression in primary GCs isolated from GC-specific paxillin knockout mice (PXN-KO GCs), which were created by crossing Amhr2 -Cre expressing mice with Pxn -floxed mice as recently reported ( Vann et al , 2023 ). Similar to KGN cells, AR protein content was slightly but statistically significantly decreased, but Ar mRNA was not affected in PXN-KO GCs ( Fig.…”

“…GC-specific paxillin knockout mice were generated as previously published using the Cre-lox system ( Vann et al , 2023 ). Briefly, paxillin-floxed mice ( Pxn fl/fl ) were crossed with mice expressing Cre recombinase driven by the anti-Müllerian hormone receptor 2 ( Amhr2 ) promoter ( Amhr2 Cre/+ ).…”

“…Offspring positive for Amhr2 -Cre and two paxillin-floxed alleles ( Amhr2 Cre/+ ; Pxn fl/fl ) were considered GC-specific paxillin knockouts, while offspring negative for Amhr2 -Cre and positive for two paxillin-floxed alleles ( Amhr2 +/+ ; Pxn fl/fl ) were designated as littermate controls. We recently characterized the reproductive effects of this knockout model ( Vann et al , 2023 ). Here, we studied the effects of paxillin on AR expression in GCs using an independent cohort of these transgenic mice.…”

“…Paxillin-knockout KGN cells were generated as previously described ( Vann et al , 2023 ). Briefly, KGN cells were transfected using jetPRIME (Polyplus, Illkirch, France) with an all-in-one non-viral vector set containing three single-guide (sg) RNAs targeting the human paxillin gene (ABM catalog #382401110591, Richmond, BC, Canada) or a non-targeting control vector.…”

“…This suggests that paxillin may module ovarian function. To explore this, we recently created a GC-specific paxillin knockout mouse model and found that paxillin loss in GCs reduces cell death and increases female fertility in advanced reproductive age ( Vann et al , 2023 ). Previously, we showed that genomic AR actions in GCs may be minimal, and that AR localization is a major determinant of its stability, as its half-life increases dramatically when AR is not available for proteasomal degradation in the cytoplasm ( Astapova et al , 2021 ).…”