Cyclic nucleotide phosphodiesterase 3A–deficient mice as a model of female infertility
Silvia Masciarelli, … , Marco Conti, Vincent Manganiello
Silvia Masciarelli, … , Marco Conti, Vincent Manganiello
Published July 15, 2004
Citation Information: J Clin Invest. 2004;114(2):196-205. https://doi.org/10.1172/JCI21804.
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Article Reproductive biology

Cyclic nucleotide phosphodiesterase 3A–deficient mice as a model of female infertility

Abstract

Since cAMP blocks meiotic maturation of mammalian and amphibian oocytes in vitro and cyclic nucleotide phosphodiesterase 3A (PDE3A) is primarily responsible for oocyte cAMP hydrolysis, we generated PDE3A-deficient mice by homologous recombination. The Pde3a–/– females were viable and ovulated a normal number of oocytes but were completely infertile, because ovulated oocytes were arrested at the germinal vesicle stage and, therefore, could not be fertilized. Pde3a–/– oocytes lacked cAMP-specific PDE activity, contained increased cAMP levels, and failed to undergo spontaneous maturation in vitro (up to 48 hours). Meiotic maturation in Pde3a–/– oocytes was restored by inhibiting protein kinase A (PKA) with adenosine-3′,5′-cyclic monophosphorothioate, Rp-isomer (Rp-cAMPS) or by injection of protein kinase inhibitor peptide (PKI) or mRNA coding for phosphatase CDC25, which confirms that increased cAMP-PKA signaling is responsible for the meiotic blockade. Pde3a–/– oocytes that underwent germinal vesicle breakdown showed activation of MPF and MAPK, completed the first meiotic division extruding a polar body, and became competent for fertilization by spermatozoa. We believe that these findings provide the first genetic evidence indicating that resumption of meiosis in vivo and in vitro requires PDE3A activity. Pde3a–/– mice represent an in vivo model where meiotic maturation and ovulation are dissociated, which underscores inhibition of oocyte maturation as a potential strategy for contraception.

Authors

Silvia Masciarelli, Kathleen Horner, Chengyu Liu, Sun Hee Park, Mary Hinckley, Steven Hockman, Taku Nedachi, Catherine Jin, Marco Conti, Vincent Manganiello

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Figure 1

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Gene and protein structures in Pde3a+/+ and Pde3a–/– mice. (A) Putative ...
Gene and protein structures in Pde3a+/+ and Pde3a–/– mice. (A) Putative gene structure of mouse Pde3a (NCBI accession number NT_039360). Double slash marks (//) indicate that introns are not drawn to scale. (B) From an approximately 7-kb genomic (129/SvJ) fragment that included MPde3a exons 11–14, a targeting vector was constructed that consisted of an approximately 2.5-kb fragment containing MPde3A exons 11 and 12, the NPTII coding sequence (Neo), an approximately 3.5-kb fragment containing MPde3a exon 14, and the TK coding sequence. By homologous recombination in 129/SvJ ES cells, MPde3a exon 13 (WT) was replaced by Neo cassette (mt). (C) Southern blots (using a probe directed upstream from the 5′ end of the genomic fragment used to construct the targeting vector) and PCR analysis of genomic DNA isolated from tails of Pde3a+/+, Pde3a+/–, or Pde3a–/– mice. Top row: BamH1 restriction fragments of approximately 12 kb and 7.5 kb from Pde3a+/+ and Pde3a–/– mice, respectively. Bottom row: PCR amplification of part of exon 13 (∼136 bp) of the normal (+) allele; and of part of Neo sequence (∼487 bp) of the mutant (–) allele. (D) Structural organization of the MPDE3A catalytic domain (AAs 714–975), depicting conserved Zn++-binding domains (AAs 752–825 and 836–866) and translated sequence of exon 13 (AAs 856–923) deleted in the Pde3a–/– genome. (E) Western blot of solubilized proteins of heart, lung, liver, and fat pads (100 μg/lane) with rabbit anti-PDE3A (top) or anti-PDE3B (bottom) IgG.