Loss of vascular endothelial growth factor A (VEGFA) isoforms in granulosa cells using pDmrt-1-Cre or Amhr2-Cre reduces fertility by arresting follicular development and by reducing litter size in female mice.

Wei Shen,Kevin M. Sargent,David W. Silversides,Debra T. Clopton,Napoleone Ferrara,Ningxia Lu,Andrea S. Cupp,Vanessa M. Brauer,William E. Pohlmeier

doi:10.1371/journal.pone.0116332

Abstract

Because VEGFA has been implicated in follicle development, the objective of this study was to determine the effects of granulosa- and germ cell-specific VEGFA loss on ovarian morphogenesis, function, and female fertility. pDmrt1-Cre mice were mated to floxed VEGFA mice to develop granulosa-/germ cell-specific knockouts (pDmrt1-Cre;Vegfa -/-). The time from mating to first parturition was increased when pDmrt1-Cre;Vegfa -/- females were mated to control males (P = 0.0008) and tended to be longer for heterozygous females (P < 0.07). Litter size was reduced for pDmrt1-Cre;Vegfa -/- females (P < 0.007). The time between the first and second parturitions was also increased for heterozygous females (P < 0.04) and tended to be increased for pDmrt1-Cre;Vegfa -/- females (P < 0.07). pDmrt1-Cre;Vegfa -/- females had smaller ovaries (P < 0.04), reduced plasma estradiol (P < 0.007), fewer developing follicles (P < 0.008) and tended to have fewer corpora lutea (P < 0.08). Expression of Igf1r was reduced (P < 0.05); expression of Foxo3a tended to be increased (P < 0.06); and both Fshr (P < 0.1) and Sirt6 tended to be reduced (P < 0.06) in pDmrt1-Cre;Vegfa -/- ovaries. To compare VEGFA knockouts, we generated Amhr2-Cre;Vegfa -/- mice that required more time from mating to first parturition (P < 0.003) with variable ovarian size. Both lines had more apoptotic granulosa cells, and vascular staining did not appear different. Taken together these data indicate that the loss of all VEGFA isoforms in granulosa/germ cells (proangiogenic and antiangiogenic) causes subfertility by arresting follicular development, resulting in reduced ovulation rate and fewer pups per litter.

Highlights

Vascular endothelial growth factor A (VEGFA) is one of the key factors regulating angiogenesis in the ovary [1]
The findings generated from the current study demonstrated that loss of VEGFA in granulosa and germ cells resulted in subfertile females
While VEGFA has been previously implicated in fertility, this is the first time conditional loss of both pro- and antiangiogenic VEGFA isoforms has been accomplished within the granulosa cells of the ovary

Summary

Introduction

Vascular endothelial growth factor A (VEGFA) is one of the key factors regulating angiogenesis in the ovary [1]. VEGFA stimulates neovascularization, vascular permeability, acts as a survival factor and can stimulate proliferation of vascular and nonvascular cells [2,3,4]. KDR is involved in mediating endothelial cell proliferation, survival and vascular permeability, whereas FLT1 may be inhibitory by sequestering VEGFA, and preventing its interaction with KDR [9]. Roberts et al reported ovarian administration of antibodies to KDR resulted in significant depletion of primordial follicle numbers, whereas anti-FLT1 antibodies did not [10]. It appears that KDR is critical for follicle progression

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