PDE3A is involved in the degradation of cAMP in the oocyte and heart muscle. Inhibitors of phosphodiesterase 3 were used to block meiosis in ovulating oocytes in rodents (Wiersma et al., 1998). By this strategy, it was demonstrated that fertilization and pregnancy could be prevented without disturbing follicle rupture and normal estrous cyclicity.
The phosphodiesterase 3 inhibitor ORG 9935 inhibits oocyte maturation in the naturally selected dominant follicle in rhesus macaques. Jensen JT et al. BACKGROUND: The study was conducted to determine whether the phosphodiesterase (PDE) 3 inhibitor ORG 9935 prevents the resumption of meiosis in primate oocytes during natural menstrual cycles. STUDY DESIGN: Regularly cycling adult female macaques (n=8) were followed during the follicular phase and then started on a 2-day treatment regimen of human recombinant gonadotropins to control the timing of ovulation. Monkeys received no further treatment (controls) or ORG 9935. Oocytes were recovered by laparoscopic follicle aspiration 27 h after an ovulatory stimulus, cultured in vitro in the absence of inhibitor and inseminated. The primary outcome was the meiotic stage of the oocyte. RESULTS: In six ORG 9935 cycles, five of the recovered oocytes were germinal vesicle (GV)-intact, and one exhibited GV breakdown (GVBD). In contrast, all three oocytes that recovered during control cycles were GVBD (p<.05). None of the ORG 9935-treated oocytes underwent fertilization compared with 2/3 (67%) from controls. CONCLUSIONS: These results demonstrate that ORG 9935 blocks resumption of meiosis in the naturally selected dominant follicle in primates and suggest that PDE3 inhibitors have potential clinical use as contraceptives in women.
NCBI Summary:
This gene encodes a member of the cGMP-inhibited cyclic nucleotide phosphodiesterase (cGI-PDE) family. cGI-PDE enzymes hydrolyze both cAMP and cGMP, and play critical roles in many cellular processes by regulating the amplitude and duration of intracellular cyclic nucleotide signals. The encoded protein mediates platelet aggregation and also plays important roles in cardiovascular function by regulating vascular smooth muscle contraction and relaxation. Inhibitors of the encoded protein may be effective in treating congestive heart failure. Alternatively spliced transcript variants encoding multiple isoforms have been observed for this gene. [provided by RefSeq, Sep 2011]
The effect of pre-maturation culture using phosphodiesterase type 3 inhibitor and insulin, transferrin and selenium on nuclear and cytoplasmic maturation of bovine oocytes. Guimarães AL et al. (2015) This study aims to evaluate if a pre-maturation culture (PMC) using cilostamide as a meiotic inhibitor in combination with insulin, transferrin and selenium (ITS) for 8 or 24 h increases in vitro embryo production. To evaluate the effects of PMC on embryo development, cleavage rate, blastocyst rate, embryo size and total cell number were determined. When cilostamide (20 μM) was used in PMC for 8 or 24 h, 98% of oocytes were maintained in germinal vesicles. Although the majority of oocytes resumed meiosis after meiotic arrest, the cleavage and blastocyst rates were lower than the control (P 0.05) to the control. The deleterious effect of 20 μM cilostamide treatment for 24 h on a PMC was confirmed by lower cumulus cell viability, determined by trypan blue staining, in that group compared with the other groups. A lower concentration (10 μM) and shorter exposure time (8 h) minimized that effect but did not improve embryo production. More studies should be performed to determine the best concentration and the arresting period to increase oocyte competence and embryo development.//////////////////
The role of cilostazol, a phosphodiesterase 3 inhibitor, on oocyte maturation and subsequent pregnancy in mice. Li M et al. It is important to identify effective contraceptive drugs that cause minimal disruption to physiological processes. Phosphodiesterase 3 (PDE3) inhibitors suppress meiosis in oocytes by decreasing the level of cAMP and blocking the extrusion of the first polar body. In this study, we tested the PDE3 inhibitor, cilostazol, as a potential contraceptive agent. The effects of cilostazol treatment in vitro and in vivo on the suppression of oocyte maturation in a mouse model were investigated. The results indicated that treatment with increasing concentrations of cilostazol led to a dose-dependent arrest in meiosis progression. The effective in vitro concentration was 1 ?M and was 300 mg/kg in vivo. The effect of cilostazol was reversible. After removal of the drug, meiosis resumed and mouse oocytes matured in vitro, and showed normal chromosome alignment and spindle organization. After fertilization using an ICSI method, the oocytes showed normal morphology, fertilization rate, embryo cleavage, blastocyst formation, and number of viable pups when compared with controls. The offspring showed similar body weight and fertility. In vivo, the mice became infertile if the drug was injected sequentially, and became pregnant following discontinuation of cilostazol. More importantly, no side effects of cilostazol were observed in treated female mice as demonstrated by blood pressure and heart rate monitoring. It is concluded that cilostazol, a drug routinely used for intermittent claudication, can effectively inhibit oocyte maturation in vitro and in vivo, does not affect the developmental potential of oocytes following drug removal and has few side effects in female mice treated with this drug. These findings suggest that cilostazol may be a potential new contraceptive agent that may facilitate an efficacy and safety study of this drug.
The phosphodiesterase 3 inhibitor ORG 9935 inhibits oocyte maturation during gonadotropin-stimulated ovarian cycles in rhesus macaques Jensen JT, et al .
To determine whether phosphodiesterase (PDE) 3 inhibitors prevent the resumption of meiosis by primate oocytes in vivo, rhesus macaques were stimulated to develop multiple preovulatory follicles by administering human recombinant gonadotropins, and follicles were aspirated 34 h after an ovulatory stimulus (human chorionic gonadotropin ). Monkeys received no further treatment (controls) or the PDE3 inhibitor ORG 9935 (a) exclusively in the periovulatory interval beginning 6-12 h prior to receiving hCG at 200 mg/kg every 12 h orally (PER200) or a 200 mg/kg oral loading dose followed by 50 mg/kg sc every 6 h (PER50) or (b) throughout the ovarian stimulation protocol with daily increases until a dose of 200 mg/kg bid was administered onward from the eighth day of ovarian stimulation (EXT200). The primary outcome was the number of oocytes that had resumed meiosis (germinal vesicle breakdown ) at collection. At initial aspiration, 85% of oocytes recovered from control animals (n=4) had progressed to GVBD compared with 53% (p<.01), 23% (p<.01), and 13% (p<.01) recovered from animals in the PER200 (n=2), PER50 (n=1) and EXT200 (n=3) groups, respectively. Although spontaneous maturation of oocytes was observed during follow-up culture in the absence of ORG 9935, none of the oocytes in the PER50 or EXT200 underwent normal fertilization in vitro. These results demonstrate that the PDE3 inhibitor ORG 9935 blocks oocyte maturation during gonadotropin-stimulated ovarian cycles in rhesus macaques and suggest that PDE3 inhibitors have potential clinical use as contraceptives in women.
Effects of long-term in vitro exposure to phosphodiesterase type-3 inhibitors on follicle and oocyte development Nogueira D, et al .
Germinal vesicle (GV)-stage oocytes retrieved from antral follicles undergo nuclear maturation in vitro, which typically occurs prior to cytoplasmic maturation. Short-term culture with meiotic inhibitors has been applied to arrest oocytes at the GV stage aiming to synchronize nuclear and ooplasmic maturity. However, the results obtained are still far from the in vivo situation. In order to acquire competence, immature oocytes may require meiotic arrest in vitro for a more extended period. The phosphodiesterase type 3-inhibitor (PDE3-I) is a potent meiotic arrester. The effects of a prolonged culture with PDE3-I on oocyte quality prior to and after reversal from the inhibition are not known. This study tested the impact of long-term in vitro exposure of two PDE3-Is, org9935 and cilostamide, on oocytes using a mouse follicle culture model. The results showed that PDE3-I (maximum of 10 muM) during a 12-day culture of follicle-enclosed oocytes did not alter somatic cell proliferation, differentiation or follicle survival. In addition, the steroid production profile was not significantly modified by a 12-day exposure to PDE3-I. The recombinant human chorionic gonadotrophin/recombinant human epidermal growth factor stimulus induced a characteristic normal progesterone peak of luteinization and normal mucification of the cumulus cells, while the enclosed oocyte remained blocked at the GV stage. In vitro maturation of denuded or cumulus-enclosed oocytes derived from org9935- or cilostamide-exposed follicles progressed through meiosis and formed morphologically normal meiotic spindles with chromosomes properly aligned at the equator. In conclusion, long-term culture with PDE3-I was harmless to somatic cell function, differentiation, oocyte growth and maturation. Our results suggested that PDE3-I can be applied when extended oocyte culture is required to improve ooplasmic maturation.
Cellular localization
Cytoplasmic
Comment
Disruption of Gap Junctional Communication within the Ovarian Follicle Induces Oocyte Maturation Sela-Abramovich S, et al .
Meiotically-arrested mammalian oocytes are stimulated to resume meiosis by LH. This response, which can be reversed by elevation of intraoocyte cAMP levels, is associated with interruption of gap junctional communication (GJC) within the ovarian follicle. In the present study, we examined the hypothesis that disruption of GJC within the ovarian follicle is sufficient for induction of oocyte maturation. For this purpose, we incubated rat follicle-enclosed oocytes (FEOs) with carbenoxolone (CBX), a known blocker of gap junctions. We found that this selective disruptor of GJC promoted maturation of almost all the FEOs after 5 h of incubation; this response was also obtained by a transient (two hour) exposure to this agent. CBX-induced oocyte maturation was accompanied by a substantial decrease in intraoocyte concentrations of cAMP that was not associated with elevated activity of type 3A phosphodiesterase (PDE3A). The effect of CBX on reinitiation of meiosis was blocked by isobutylmethylxanthine (IBMX), a phosphodiesterase inhibitor. Unlike LH, CBX did not activate MAPK in the follicular cells, and inhibition of the MAPK signaling pathway by means of UO126, did not prevent the resumption of meiosis. Injection of CBX into the ovarian bursa of intact animals stimulated maturation in 30% of the oocytes, whereas no maturation was observed in the contralateral ovary injected with PBS. We conclude that since experimentally-induced breakdown of communication within the ovarian follicle is associated with a drop in intraoocyte cAMP concentrations and results in resumption of meiosis, this could be the physiological mechanism employed by LH to stimulate oocyte maturation.
Ovarian function
Oogenesis, Oocyte maturation, Early embryo development
Comment
High cGMP and low PDE3A activity are associated with oocyte meiotic incompetence. Gershon E et al. (2019) Resumption of meiosis in mammalian oocytes, defined as oocyte maturation, is stimulated by luteinizing hormone (LH). Fully grown oocytes can also mature spontaneously, upon their release from the ovarian follicle. However, growing oocytes fail to resume meiosis in vitro and the mechanism underlying their meiotic incompetence is unknown. It is commonly accepted that a drop in intraoocyte cyclic guanosine monophosphate (cGMP) resulting in the elevated activity of the oocyte-specific PDE3A leads to a decrease in cAMP content, essential for reinitiation of meiosis. We explored the regulation of these cyclic nucleotides and their degrading PDE3A in growing oocytes. Our research addressed the LH-induced rather than spontaneous oocyte maturation. We examined 16-21 as compared to 25-day-old, PMSG-primed rats, treated with the LH analog, hCG. The effect of LH was also examined ex vivo, in isolated ovarian follicles. We found that hCG failed to induce oocyte maturation and ovulation in the younger animals and that ovulation-associated genes were not upregulated in response to this gonadotropin. Furthemore, the drop of intraoocyte cGMP and cAMP observed in fully grown oocytes upon exposure of the ovary to LH, was not detected in growing oocytes. Interestingly, whereas the global expression of PDE3A in growing and fully grown oocytes is similar, a significantly lower activity of this enzyme was determined in growing oocytes. Our findings show that meiotic incompetence is associated with a relatively high oocyte cGMP concentration and a low activity of PDE3A, which in follicle-enclosed oocytes may represent the failure of the somatic follicle cells to respond to LH.//////////////////
Cilostazol blocks pregnancy in naturally cycling swine: An animal model. Taiyeb AM et al. (2015) Cilostazol (CLZ) is an FDA approved therapeutic that is indicated for patients with intermittent claudication disease. CLZ is a selective inhibitor for phosphodiesterase 3A (PDE3A); an enzyme that controls oocyte maturation in many mammals including humans. Recently, CLZ has been reported to block pregnancy and oocyte maturation in mice. The objective of the present work was to evaluate the potential non-steroidal contraceptive capacity of CLZ using a more advanced translational model for humans. Three groups of naturally cycling sows were treated orally with 0, 100, or 200mg CLZ, twice a day (bid), for 6days before estrus and continued for three days after estrus. Each sow was mated by one of two proven fertile boars on alternate days during estrus. CLZ dose of 100mg, bid, completely blocked pregnancy in sows when compared to control sows (P<0.01). However, the 200mg dose of CLZ, bid, failed to significantly block pregnancy in pigs. No significant differences were observed in heart rates of treated and control animals. Re-mating of the previously treated sows exhibited normal pregnancies and litter sizes. This study shows that CLZ is capable of producing a reversible non-steroidal contraceptive effect without adverse effects on the heart rate in pigs. The observed contraceptive effect of CLZ was at doses similar to those indicated to humans. This FDA approved agent, for treatment of patients with intermittent claudication, may have an additional therapeutic effect as a non-steroidal contraceptive agent. Cilostazol merits further evaluation in women.//////////////////
Phosphodiesterase 3 (PDE3) inhibition with Cilostazol does not block in vivo oocyte maturation in rhesus macaques (Macaca mulatta). Hanna CB et al. (2015) Studies in mice suggest that cilostazol, an FDA-approved phosphodiesterase 3 (PDE3) inhibitor, might have a contraceptive effect within the approved dose range. We sought to evaluate the potential contraceptive effects of cilostazol in a non-human primate model. Adult female rhesus macaques were stimulated to develop multiple preovulatory follicles by administering human recombinant gonadotropins and oocytes were collected by follicle aspiration 36hours after an ovulatory stimulus (human chorionic gonadotropin; hCG). Monkeys received no further treatment (controls), or the PDE3 inhibitor cilostazol at the maximum approved human dose of 100mg twice daily starting 6days prior to follicle aspiration. Recovered oocytes were scored for meiotic stage germinal vesicle (GV) intact, germinal vesicle breakdown (GVDB/], and metaphase II stage oocyte were fertilized in vitro and observed for normal embryo development. Similar proportions of GV stage oocytes were recovered from control (27%±4) and cilostazol (27%±9) treated females, and the proportion of embryos that developed into blastocysts was also similar for both groups (7%±5 control vs 15%±8 cilostazol). Oral dosing of cilostazol tablets during controlled ovarian stimulation protocols did not prevent oocyte maturation or embryo development in macaques. Since administration of the maximum approved human dose of cilostazol (an FDA-approved PDE3 inhibitor) to macaques did not prevent oocyte maturation or fertilization, it is not likely that this dose would be contraceptive in women.//////////////////
Simulated physiological oocyte maturation (SPOM): a novel in vitro maturation system that substantially improves embryo yield and pregnancy outcomes. Albuz FK et al. Oocyte in vitro maturation (IVM) reduces the need for gonadotrophin-induced ovarian hyperstimulation and its associated health risks but the unacceptably low conception/pregnancy rates have limited its clinical uptake. We report the development of a novel in vitro simulated physiological oocyte maturation (SPOM) system.
The role of cilostazol, a phosphodiesterase 3 inhibitor, on oocyte maturation and subsequent pregnancy in mice. Li M et al. It is important to identify effective contraceptive drugs that cause minimal disruption to physiological processes. Phosphodiesterase 3 (PDE3) inhibitors suppress meiosis in oocytes by decreasing the level of cAMP and blocking the extrusion of the first polar body. In this study, we tested the PDE3 inhibitor, cilostazol, as a potential contraceptive agent. The effects of cilostazol treatment in vitro and in vivo on the suppression of oocyte maturation in a mouse model were investigated. The results indicated that treatment with increasing concentrations of cilostazol led to a dose-dependent arrest in meiosis progression. The effective in vitro concentration was 1 ?M and was 300 mg/kg in vivo. The effect of cilostazol was reversible. After removal of the drug, meiosis resumed and mouse oocytes matured in vitro, and showed normal chromosome alignment and spindle organization. After fertilization using an ICSI method, the oocytes showed normal morphology, fertilization rate, embryo cleavage, blastocyst formation, and number of viable pups when compared with controls. The offspring showed similar body weight and fertility. In vivo, the mice became infertile if the drug was injected sequentially, and became pregnant following discontinuation of cilostazol. More importantly, no side effects of cilostazol were observed in treated female mice as demonstrated by blood pressure and heart rate monitoring. It is concluded that cilostazol, a drug routinely used for intermittent claudication, can effectively inhibit oocyte maturation in vitro and in vivo, does not affect the developmental potential of oocytes following drug removal and has few side effects in female mice treated with this drug. These findings suggest that cilostazol may be a potential new contraceptive agent that may facilitate an efficacy and safety study of this drug.
In isolated oocytes, spontaneous GVBD was blocked by two inhibitors of type 3 PDE (cGMP-inhibited: CGI-PDE), milrinone and cilostamide (Tsafiri et al., 1996). Furthermore, inhibitors of phosphodiesterase blocked rodent oocyte meiosis in vivo (Wiersma et al., 1998).
Phosphodiesterase 3 inhibitors selectively block the spontaneous resumption of meiosis by macaque oocytes in vitro Jensen JT,et al .
The purpose of this study was to determine whether phosphodiesterase (PDE) 3 inhibitors selectively prevent the resumption of meiosis in primates. METHODS: Immature oocytes (intact germinal vesicles) obtained from large pre-ovulatory follicles following ovarian stimulation in rhesus macaques were incubated with or without various doses of the PDE3 inhibitors, Cilostamide, Milrinone or ORG 9935, or a selective PDE4 inhibitor, Rolipram. Oocytes were observed for germinal vesicle breakdown (GVBD) as an indicator of resumption of meiosis. RESULTS: At 24 h, 72 of 121 (60%) control oocytes progressed to GVBD compared with 9/34 (27%, P < 0.01), 4/36 (11.1%, P < 0.01) and 0/28 (0%, P < 0.01) oocytes incubated with ORG 9935 at 0.1, 0.5 and 1.0 micromol/l respectively. Similar results were achieved at 24 h with 1.0 micromol/l Cilostamide (2/24 oocytes, 8%, P < 0.01) and 100 micromol/l Milrinone (2/32, 6%, P < 0.01). In contrast, no significant difference in GVBD was noted between control oocytes and those incubated with up to 100 micromol/l Rolipram for 24 h (43/58, 74%) or 48 h (44/58, 76%). CONCLUSIONS: These experiments establish the specificity and dose-dependent ability of PDE3, but not PDE4, inhibitors to block resumption of meiosis in macaque oocytes in vitro. Thus, PDE3 inhibitors have potential use as contraceptives in primates.
Nogueira D,et al reported that human Oocytes Reversibly Arrested in Prophase I by Phosphodiesterase Type 3 Inhibitor In Vitro.
Presence of phosphodiesterase type 3 A (PDE 3A) mRNA was confirmed in human germinal vesicle-stage (GV) oocytes. Making use of a selective PDE 3 inhibitor, Org 9935 (10 microM), oocytes retrieved from immature follicles were arrested in prophase I with a high efficiency for up to 72 h. Cumulus oocyte complexes (COCs) were retrieved in the follicular phase of the cycle before or after exposure to endogenous LH or hCG administration in vivo and randomly distributed into maturation medium with or without the PDE 3 inhibitor. Previous exposure of small follicles to LH activity in vivo had no influence on the arresting capacity of the PDE 3 inhibitor. Reversal from pharmacological arrest leads to a progression through meiosis in a normal time frame with formation of a well-aligned metaphase plate. Ultrastructure analysis of COC derived from follicles between 8 and 12 mm showed that the induced extension of prophase I arrest in vitro resulted in cytoplasm changes but not in apparent nuclear changes during culture.
Thomas RE, et al reported the effect of Specific Phosphodiesterase Isoenzyme Inhibitors During In Vitro Maturation of Bovine Oocytes on Meiotic and Developmental Capacity.
Compared to oocytes matured in vivo, in vitro matured (IVM) oocytes are compromised with respect to their capacity to support early embryo development. Delaying spontaneous in vitro meiotic maturation using specific phosphodiesterase (PDE) isoenzyme inhibitors may permit more complete oocyte cytoplasmic maturation, possibly by prolonging cumulus cell (CC)-oocyte gap junctional communication during meiotic resumption. This study aimed to investigate the effect of the isoenzyme 3 (oocyte) and 4 (granulosa cell) specific phosphodiesterase inhibitors on the kinetics of IVM and on subsequent oocyte developmental competence. Cumulus-oocyte complexes from antral bovine follicles were isolated and cultured in the presence of the specific PDE inhibitors milrinone (type 3) or rolipram (type 4) (100 micro M). In the presence of FSH, both PDE inhibitors only slightly extended CC-oocyte gap junctional communication over the first 9h, but completely blocked meiotic resumption during this period (P<0.001). The indefinite inhibitory effect of milrinone on meiotic resumption (30% GV after 48h) was overridden by 24 h when treated with FSH, but not hCG, suggesting a form of induced meiotic resumption. Oocytes treated with FSH with or without either PDE inhibitor were inseminated at either 24, 26, or 28 h. Treated with either the type 3 or type 4 phosphodiesterase inhibitor significantly (P<0.05) increased embryo development to the blastocyst stage by 33-39% (to average of 52% blastocysts) compared to control oocytes (38%) after insemination at 28h, and significantly (P<0.05) increased blastocyst cell numbers when inseminated at 24h. These results suggest that delayed spontaneous meiotic maturation, coupled with extended gap junctional communication between the cumulus cells and the oocyte has a positive effect on oocyte cytoplasmic maturation, thereby improving oocyte developmental potential.
Meiotic Arrest In Vitro by Phosphodiesterase 3-Inhibitor Enhances Maturation Capacity of Human Oocytes and Allows Subsequent Embryonic Development Nogueira D, et al .
Controlling nuclear maturation during oocyte culture might improve nuclear-cytoplasmic maturation synchrony. We aimed to evaluate the quality of in-vitro matured germinal-vesicle (GV)-stage human oocytes following a pre-maturation culture (PMC) with a meiotic arrester, phosphodiesterase 3-inhibitor (PDE3-I). Follicles (6-12 mm) were retrieved 34-36 h post-hCG administration from informed, consenting patients who had undergone controlled ovarian stimulation. Cumulus-enclosed oocytes (CEO) presenting moderate expansion or full compaction were placed in PMC with the PDE3-I, Org9935, for 24 h or 48 h. Subsequently, oocytes were removed from PMC, denuded of cumulus cells, matured in-vitro, fertilized and resulting embryos cultured. In the presence of PDE3-I ~98% of the oocytes were arrested at GV-stage. Following PDE3-I removal, oocytes acquired a higher maturation rate than oocytes that were immediately in vitro matured denuded of cumulus cells after retrieval (67% versus 46%, P = 0.01). In controls, immature CEO retrieved with moderate expansion reached higher maturation rates than fullycompacted CEO, but in PMC groups high values of maturation were achieved for both morphological classes of CEO. There was no effect of PMC on fertilization. A 24h-PMC period proved most effective in preserving embryonic integrity. Similar proportions of nuclear abnormalities were observed in embryos of all in-vitro groups. In summary, PMC with the specific-PDE3-I had a beneficial effect on human CEO by enhancing maturation, benefiting mainly fully-compacted CEO. This resulted in increased yield of mature oocytes available for insemination, without compromising embryonic development. These results suggest that applying an inhibitor to control the rate of nuclear maturity by regulating intra-oocyte PDE3 activity may allow the synchronization of nuclear and ooplasmic maturation.
Effect of a phosphodiesterase type 3 inhibitor in oocyte maturation medium on subsequent mouse embryo development. Jee BC et al. OBJECTIVE: To investigate the effect of a phosphodiesterase type 3 inhibitor in oocyte maturation medium on subsequent embryo development. DESIGN: Animal study. SETTING: University laboratory. ANIMAL(S): CD-1 mice. INTERVENTION(S): Immature oocytes were arrested meiotically in medium containing a specific phosphodiesterase type 3 inhibitor, cilostamide (1 mumol/L, 5 mumol/L, and 10 mumol/L), for 6 and 24 hours of prematuration culture, and then matured in vitro in inhibitor-free medium. MAIN OUTCOME MEASURE(S): Oocyte maturation, fertilization, and early embryonic development in vitro. RESULT(S): After 6 and 24 hours of prematuration culture with 1 mumol/L, 5 mumol/L, and 10 mumol/L cilostamide, there were no differences in maturation, fertilization, and early embryonic development compared with non-arrested controls. CONCLUSION(S): Prematuration culture with cilostamide, for either 6 or 24 hours, may not be beneficial for subsequent embryonic development of immature mouse oocytes.
Cilostazol blocks pregnancy in naturally cycling mice. Albarzanchi AM et al. BACKGROUND: Administration of a phosphodiesterase three enzyme inhibitor (PDE3-I) in rodents and primates results in ovulation of immature oocytes. Concerns regarding inhibition of PDE3 enzymes that are expressed in heart and blood vessels discouraged further development of PDE3-Is as nonsteroidal contraceptives. Cilostazol (CLZ) is a PDE3A-I that is approved for medical indications in humans and has an additional effect of adenosine uptake inhibition that is believed to counterbalance the undesirable outcomes resulting from PDE inhibition. STUDY DESIGN: Cycling mature female mice were treated with 7.5 or 15 mg CLZ, dimethyl sulfoxide or water beginning on the day of proestrus. Animals were placed with fertility-proven males after 3 days of treatment. Treatments were continued until 1 day after detection of a vaginal plug, and then females were monitored up to 30 days postbreeding to assess the effects of the compounds on pregnancy. Each of the treated female with CLZ was then remated with the same male and again monitored up to 30 days. RESULTS: None of the CLZ-treated mice produced offspring, whereas all of the control animals maintained pregnancy and delivered normal pups (p<.0001). Remating of the previously CLZ-treated females exhibited normal pregnancies and gave birth to live offspring that were not different from the controls. CONCLUSION: CLZ is a potential nonsteroidal contraceptive agent that merits further evaluation in other mammals.
Expression regulated by
LH
Comment
3'5'-Cyclic adenosine monophosphate-dependent upregulation of Pde3a in porcine cumulus cells. Sasseville M et al. The means by which cumulus cells react to gonadotropin stimulation and regulate the subsequent production and degradation of cAMP are largely unknown. In this article we report that cyclic nucleotide phosphodiesterase type 3A (Pde3a) is transcriptionally regulated in porcine cumulus cells by a cAMP-dependent pathway during in vitro maturation (IVM). Cyclic AMP-PDE activity was increased in the cumulus-oocyte complex (COC) after 10 hours of IVM and 78% of this increase was sensitive to a Pde3-specific inhibitor, cilostamide. While no variation was observed in the oocyte, cilostamide-sensitive cAMP-PDE activity increased in the cumulus cells after IVM. This was supported by Western blotting, which showed that the intensity of a 135 kDa anti-Pde3a immunoreactive band was increased in COC after IVM. The Pde3a mRNA level was upregulated 28-fold in the COC after 4 hours of IVM and remained high up to 12 hours. The mRNA upregulation and increased activity were inhibited by an RNA synthesis inhibitor, alpha-amanitin. The cilostamide-sensitive increase in PDE activity was inhibited by a protein synthesis inhibitor, cycloheximide. Pregnant mare serum gonadotropin (PMSG) caused dose-dependent activation of Pde3. The PMSG-dependent increase in Pde3 activity and Pde3a mRNA were mimicked by the adenylyl cyclase activator forskolin or prostaglandin E2. PMSG-dependent Pde3 activation was inhibited by the PKA-specific inhibitor, H89. Collectively, our results show for the first time that degradation of the intracellular cyclic nucleotide by PDE3A is transcriptionally upregulated via a cAMP-dependent pathway in cumulus cells, suggesting that it has a functional role during the ovulatory gonadotropin surge.
Role of phosphodiesterase type 3A in rat oocyte maturation. Richard FJ et al. It is generally accepted that cyclic nucleotides are key signaling molecules in the control of oocyte meiotic resumption. Given the role of phosphodiesterases (PDEs) in cyclic nucleotide degradation, this study was undertaken to investigate the properties and regulation of PDEs expressed in rat oocytes. Cilostamide-sensitive PDE3 was the major activity detected in denuded oocytes, whereas no PDE3 activity could be detected in cumulus cells. Moreover, comparable levels of PDE3 activity were measured in cumulus-oocyte complexes (COCs) and in denuded oocytes. The oocyte PDE was recovered in the soluble fraction of the homogenate and immunoprecipitated with a specific PDE3A antibody. A significant and transient increase (P < 0.05) in PDE3 activity was measured in the oocytes after 30 min of culture (70 min after isolation) compared with immediately after collection (10 min after isolation). Conversely, no changes in activity were observed when denuded oocytes or cumulus cells were incubated for up to 130 min. Evaluation of oocyte maturation indicated that only 10% of oocytes had resumed meiosis at the peak of the PDE3 activity. A significant increase (P < 0.05) in PDE3 activity was measured in COCs when follicle-enclosed oocytes were cultured in the presence of hCG. Again, this increase preceded oocyte maturation. In conclusion, these data demonstrate that PDE3A is the major PDE form expressed in mammalian oocytes. PDE3A activity increases prior to resumption of meiosis in both spontaneous and gonadotropin-stimulated maturation. These findings strongly support the hypothesis that an increase in oocyte PDE3A activity is one of the intraoocyte mechanisms controlling resumption of meiosis in rat oocytes, at least in vitro.
Ovarian localization
Oocyte, Cumulus
Comment
Shitsukawa K, et al reportd the cloning and Characterization of the Cyclic Guanosine
Monophosphate-Inhibited Phosphodiesterase PDE3A
Expressed in Mouse Oocyte.
Polymerase chain reactions using a
mouse oocyte cDNA library as a template, and primers based on the
conserved sequence of rat and human PDE3As, yielded partial fragments
corresponding to mouse PDE3A. Further screening of the mouse oocyte
cDNA library and subsequent ligation of individual cDNA clones yielded
PDE3A cDNA containing the entire coding region of mouse PDE3A. To
determine the kinetic properties of this PDE, the cDNAs encoding the
full-length PDE3A and NH(2)-truncation forms Delta 1 (Delta346aa) and
Delta 2 (Delta608aa) were expressed in mouse Leydig tumor cells. Whereas
the full-length recombinant protein was always found in the particulate
fraction, the Delta 1 and Delta 2 truncated PDE3As were recovered mostly
in the soluble fraction. The Michaelis constant values for hydrolysis of cAMP
of PDE3A Delta 1 and PDE3A Delta 2 were similar to those of intact
full-length PDE3A or oocyte PDE (0.2-0.5 &mgr;M). More importantly,
there was good correlation between the rank of potency of selective and
nonselective compounds in inhibiting recombinant PDE3A or PDE activity
derived from cumulus-oocyte complexes and in blocking resumption of
meiosis. These data provide evidence that the PDE expressed in the oocyte is
a soluble form of PDE3A and that activity of this enzyme is involved in the
control of resumption of meiosis.
Follicle stages
Secondary, Antral, Preovulatory
Comment
Effect of specific phosphodiesterase isoenzyme inhibitors during in vitro maturation of bovine oocytes on meiotic and developmental capacity.
Thomas RE, et al .
Compared with oocytes matured in vivo, in vitro-matured oocytes are compromised in their capacity to support early embryo development. Delaying spontaneous in vitro meiotic maturation using specific phosphodiesterase (PDE) isoenzyme inhibitors may permit more complete oocyte cytoplasmic maturation, possibly by prolonging cumulus cell (CC)-oocyte gap junctional communication during meiotic resumption. This study aimed to investigate the effect of the isoenzyme 3- (oocyte) and isoenzyme 4- (granulosa cell) specific PDE inhibitors on the kinetics of in vitro maturation and on subsequent oocyte developmental competence. Cumulus-oocyte complexes from antral bovine follicles were isolated and cultured in the presence of the specific PDE inhibitors milrinone (type 3) or rolipram (type 4) (100 microM). In the presence of FSH, both PDE inhibitors only slightly extended CC-oocyte gap junctional communication over the first 9 h, but they completely blocked meiotic resumption during this period (P < 0.001). The indefinite inhibitory effect of milrinone on meiotic resumption (30% at germinal vesicle stage after 48 h) was overridden by 24 h when treated with FSH, but not with hCG, suggesting a form of induced meiotic resumption. Oocytes treated with FSH with or without either PDE inhibitor were inseminated at either 24, 26, or 28 h. Treated with either the type 3 or type 4 PDE inhibitor significantly (P < 0.05) increased embryo development to the blastocyst stage by 33%-39% (to an average of 52% blastocysts) compared with control oocytes (38%) after insemination at 28 h, and significantly (P < 0.05) increased blastocyst cell numbers when inseminated at 24 h. These results suggest that delayed spontaneous meiotic maturation, coupled with extended gap junctional communication between the CCs and the oocyte has a positive effect on oocyte cytoplasmic maturation, thereby improving oocyte developmental potential.
Phenotypes
Mutations
2 mutations
Species: mouse
Mutation name: None
type: null mutation fertility: infertile - ovarian defect Comment: Cyclic nucleotide phosphodiesterase 3A-deficient mice as a model of female infertility.
Masciarelli S, et al .
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.
Species: mouse
Mutation name: None
type: null mutation fertility: infertile - ovarian defect Comment: Female infertility in PDE3A-/- mice: Polo-like kinase 1 (Plk1) may be a target of Protein Kinase A (PKA) and involved in meiotic arrest of oocytes from PDE3A-/- mice. Shen W et al. Mechanisms of cAMP/PKA-induced meiotic arrest in oocytes are not completely identified. In cultured, G2/M-arrested PDE3A (-/-) murine oocytes, elevated PKA activity was associated with inactivation of Cdc2 and Plk1, and inhibition of phosphorylation of histone H3 (S10) and of dephosphorylation of Cdc25B (S323) and Cdc2 (Thr14/Tyr15). In cultured WT oocytes, PKA activity was transiently reduced and then increased to that observed in PDE3A (-/-) oocytes; Cdc2 and Plk1 were activated, phosphorylation of histone H3 (S10) and dephosphorylation of Cdc25B (S323) and Cdc2 (Thr14/Tyr15) were observed.? In WT oocytes, PKAc were rapidly translocated into nucleus, and then to the spindle apparatus, but in PDE3A (-/-) oocytes, PKAc remained in the cytosol. Plk1 was reactivated by incubation of PDE3A (-/-) oocytes with PKA inhibitor, Rp-cAMPS. PDE3A was co-localized with Plk1 in WT oocytes, and co-immunoprecipitated with Plk1 in WT ovary and Hela cells. PKAc phosphorylated rPlk1 and Hela cell Plk1 and inhibited Plk1 activity in vitro. Our results suggest that PKA-induced inhibition of Plk1 may be critical in oocyte meiotic arrest and female infertility in PDE3A (-/-) mice.