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 5

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Regulation of maturation of cultured Pde3a+/+ and Pde3a–/– ooctyes.(A–C)...
Regulation of maturation of cultured Pde3a+/+ and Pde3a–/– ooctyes.(A–C) Time course of spontaneous maturation. (A) Denuded Pde3a+/+, not Pde3a–/–, oocytes undergo spontaneous maturation (demonstrated by GVBD) within 3 hours in vitro. (B and C) After 20 hours, Pde3a+/+ (B), but not Pde3a–/– (C), oocytes exhibit GVBD and PBs. (D) Absence of MAPK and MPF activation in Pde3a–/– oocytes. MPF and MAPK, assayed at indicated times as described in Methods, were activated (18 h) in Pde3a+/+, but not Pde3a–/–, oocytes. (E and F) Effects of PKA inhibitors on maturation of Pde3a+/+ and Pde3a–/– oocytes. (E) Pde3a+/+ (with 10 μM cilostamide) and Pde3a–/– oocytes were incubated for 24 hours without or with 5 mM Rp-cAMPS before evaluation of GVBD. In the absence of Rp-cAMPS, spontaneous maturation was blocked in both Pde3a–/– and cilostamide-treated Pde3a+/+ oocytes. GVBD and meiosis were reinitiated to the same extent by Rp-cAMPS in Pde3a–/– oocytes and cilostamide-treated Pde3a+/+ oocytes. (F) Pde3a–/– and Pde3a+/+ oocytes were incubated for the indicated times with 5 mM Rp-8-Br-cAMPS, washed twice in a drop (50 μl) of maturation medium, and incubated without Rp-8-Br-cAMPS. After 24 hours, all oocytes were assessed for GVBD and PB formation. “Total oocytes” indicates the numbers incubated under each condition. Within 1 hour of treatment of Pde3a–/– oocytes with Rp-8-Br-cAMPS, meiosis was reinitiated, as monitored by the percent decrease in GV, and increase in GVBD and PB. Data at 0, 1, 3, and 24 hours represent mean percent ± one half of the range, in two separate experiments.