Lack of functional pregnancy-associated plasma protein-A (PAPPA) compromises mouse ovarian steroidogenesis and female fertility

Abstract

The insulin-like growth factor (IGF) system plays an important role in regulating ovarian follicular development and steroidogenesis. IGF binding proteins (IGFBP) mostly inhibit IGF actions, and IGFBP proteolysis is a major mechanism for regulating IGF bioavailability. Pregnancy-associated plasma protein-A (PAPPA) is a secreted metalloprotease responsible for cleavage of IGFBP4 in the ovary. The aim of this study was to investigate whether PAPPA plays a role in regulating ovarian functions and female fertility by comparing the reproductive phenotype of wild-type (WT) mice with mice heterozygous or homozygous for a targeted Pappa gene deletion (heterozygous and PAPP-A knockout [KO] mice, respectively). When mated with WT males, PAPP-A KO females demonstrated an overall reduction in average litter size. PAPP-A KO mice had a reduced number of ovulated oocytes, lower serum estradiol levels following equine chorionic gonadotropin administration, lower serum progesterone levels after human chorionic gonadotropin injection, and reduced expression of ovarian steroidogenic enzyme genes, compared to WT controls. In PAPP-A KO mice, inhibitory IGFBP2, IGFBP3, and IGFBP4 ovarian gene expression was reduced postgonadotropin stimulation, suggesting some compensation within the ovarian IGF system. Expression levels of follicle-stimulating hormone receptor, luteinizing hormone receptor, and genes required for cumulus expansion were not affected. Analysis of preovulatory follicular fluid showed complete loss of IGFBP4 proteolytic activity in PAPP-A KO mice, demonstrating no compensation for loss of PAPPA proteolytic activity by other IGFBP proteases in vivo in the mouse ovary. Taken together, these data demonstrate an important role of PAPPA in modulating ovarian function and female fertility by control of the bioavailability of ovarian IGF.

FIG. 1.

Characterization of PAPP-A KO reproductive potential. A) Average litter size (+ SEM) from WT (black bar), HET (gray bar) and PAPP-A KO (white bar) female mice mated with WT males, determined from 10–20 litters per genotype. The average litter size in PAPP-A KO females is significantly reduced vs. WT female mice. **P < 0.01 vs. WT. B) Number of ovulated COCs (+ SEM) after induction of superovulation in 3- and 6-wk-old mice. PAPP-A KO (white bars) ovulate a significantly lower number of COCs compared to WT or HET mice (gray bars). Each bar represents between three and seven mice. *P < 0.05 vs. WT. C) Ovary weight (mg) (+ SEM) in 22-day-old mice determined 47 h after eCG priming and 4 h and 8 h after hCG injection. *P < 0.05 and **P < 0.01 compared to same genotype at 47 h eCG (0 h hCG). D) Ovary (in mg) to body weight (in g) ratio (+ SEM) in 22-day-old mice determined 47 h after eCG priming and 4 and 8 h after hCG injection. *P < 0.05 vs. WT or HET mice. E) Representative micrograph of ovarian histology in WT (left panel), HET (middle panel), and PAPP-A KO (right panel) mice. Ovaries from 22-day-old eCG-primed mice 8 h after hCG injection were fixed overnight in Bouin solution, paraffin embedded, sectioned at 5 μm, and stained with H&E. One intact ovary cross section is seen in each micrograph.

FIG. 2.

Serum steroid levels. Serum steroid E₂ (A) and P₄ (B) levels in WT (black bars), HET (gray bars), and PAPP-A KO (white bars) mice determined at indicated times after hCG injection 47 h following eCG priming. Each data point represents the average of between four and fourteen mice (+ SEM). *P < 0.05 and **P < 0.01 vs. WT.

FIG. 3.

Ovarian expression levels of genes involved in P₄ synthesis, Star (A), Cyp11a1 (B), and Hsd3b1 (C), following gonadotropin stimulation. The indicated time points are after eCG priming or after hCG injection (given 47 h after eCG). Data are from WT (black bars), HET (gray bars), and PAPP-A KO (white bars) mice. Expression levels are shown as fold change compared to WT level at 0 h (+ SEM). Each data point represents the average of between four and fourteen samples. *P < 0.05 and **P < 0.01 vs. WT.

FIG. 4.

Ovarian expression levels of E₂ synthesizing genes, Cyp17a1 (A), Cyp19a1 (B), and Hsd17b1 (C), following gonadotropin stimulation. The indicated time points are after eCG priming or after hCG injection (given 47 h after eCG). Data are from WT (black bars), HET (gray bars), and PAPP-A KO (white bars) mice. Expression levels are shown as fold change compared to WT level at 0 h (+ SEM). Each data point represents the average of between four and fourteen samples. *P < 0.05 and **P < 0.01 vs. WT.

FIG. 5.

Ovarian Pappa mRNA levels and IGFBP4 protease activity. A) Pappa mRNA levels following gonadotropin stimulation in WT (black bars), HET (gray bars), and PAPP-A KO (white bars) mice. The indicated time points are after eCG priming or after hCG injection (given 47 h after eCG). Each data point represents the average of between four and fourteen samples. *P < 0.05 and **P < 0.01 vs. unstimulated mice. B) IGFBP4 proteolytic activity in follicular fluid from WT and PAPP-A KO mice. SDS-PAGE separation of ¹²⁵I-labeled IGFBP4 incubated with medium from HEK293 cells transfected with an expression plasmid encoding recombinant human PAPPA (lanes a, b) or an empty plasmid (lane g), or follicular fluid from eCG-stimulated ovaries from WT (lanes c, d) or PAPP-A KO (lanes e, f) mice, in the presence or absence of IGF-II (indicated by - or +, respectively). The top bands represent intact (i) IGFBP4, bottom bands the cleaved (c) form of IGFBP4.

FIG. 6.

Ovarian expression levels of IGF system genes, Igfbp2 (A), Igfbp3 (B), and Igfbp4 (C). The indicated time points are after eCG priming or after hCG injection (given 47 h after eCG). Data are from WT (black bars), HET (gray bars), and PAPP-A KO (white bars) mice. Expression levels are shown as fold change compared to WT level at 0 h (+ SEM). Each data point represents the average of between four and fourteen samples. *P < 0.05 and **P < 0.01 vs. WT.

FIG. 7.

Ovarian expression of genes required for normal cumulus expansion following hCG injection, Ptgs2 (A), Has2 (B), and Tnfai6p (C). Data are from WT (black bars), HET (gray bars), and PAPP-A KO (white bars) mice. Expression levels are shown as fold change compared to WT level at 0 h hCG (47 h eCG) (+ SEM).