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Serono Reproductive Biology Institute (S.D.M., M.P., A.C., D.F., D.K., B.B., S.P.), Rockland, Massachusetts 02370; Istituto di Ricerche Biomediche "A Marxer" (E.G.T.), LCG Bioscience, 10010 Colleretto Giacosa, Italy; and Department of Obstetrics, Gynecology, and Reproductive Sciences (P.J.C.), College of Medicine, University of Saskatchewan, Saskatoon, Saskatchewan, Canada S7N 0W8
Address all correspondence and requests for reprints to: Sean D. McKenna, Serono Reproductive Biology Institute, One Technology Place, Rockland, Massachusetts 02370. E-mail: sean.mckenna{at}serono.com.
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Introduction |
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Although recent reports have identified other potential signaling pathways for the two gonadotropins (3, 5), cAMP is recognized as the primary second messenger for gonadotropin action because increasing cAMP alone is sufficient to mediate many of the gonadotropin-associated effects in vitro, including estrogen production from granulosa cells, oocyte maturation, and germinal vesicle breakdown (6). Whereas the nature of the gonadotropin-induced signaling events that determine which cellular responses are induced remain unclear, the divergence of such responses are believed to be controlled by coactivation of auxiliary signal transduction pathways, compartmentalization of regulatory components, influence of cell differentiation state, and/or qualitative differences in the cAMP signal itself (3, 5).
Phosphodiesterases (PDEs) are a family of enzymes that regulate signal transduction events that use cyclic nucleotides as second messengers by catalyzing their hydrolysis into biologically inactive 5'-nucleotide monophosphate analogs. To date, at least 11 different PDE family members have been identified, each one having multiple subtypes encoded by unique genes (7). Murine ovarian follicles express multiple forms of PDEs, including at least two subtypes of PDE4, whereas the oocyte itself expresses PDE3 (8). Several lines of evidence indicate that one or more of the PDE4 subtypes expressed in follicles are responsible for regulating the biological activity of gonadotropins in granulosa cells. Notably, PDE4D mRNA expression is up-regulated in granulosa cells after treatment with both FSH and LH (9). Hence, lack of PDE4 function may significantly impact gonadotropin-induced responses in vivo. Here we demonstrate, using pharmacological models, that selective inhibitors of PDE4 can stimulate ovulation.
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Materials and Methods |
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cAMP production in human LHR-expressing JC-410 granulosa cells
JC-410, porcine granulosa cells lacking functional endogenous gonadotropin receptors (13, 14), were maintained in DMEM/F12 supplemented with 5% newborn calf serum (Life Technologies, Inc., Grand Island, NY) and 5 µg/ml of insulin (Life Technologies, Inc.). The human LHR coding sequence was subcloned into pN
, a mammalian expression vector containing the mouse metallothionein promoter and a simian virus 40 enhancer/early region selection cassette. The construct sequence was verified with the ThermoSequenase radiolabeled terminator cycle sequencing kit (Amersham Biosciences, Piscataway, NJ). Stable cell lines were established by transfecting the LHR plasmid into JC-410 cells using Effectene (Qiagen, Valencia, CA) according to the manufacturer’s recommended DNA to lipid ratio. The cells were allowed to recover for 48 h before the monolayer was trypsinized and replated in culture medium supplemented with 300 µg/ml Geneticin (Life Technologies) for selection. The cells were routinely maintained in 300 µg/ml Geneticin. For cAMP determinations, the cells were plated at a density of 25,000 cells/well in 96-well plates 1 d before the assay. The following day, the cells were stimulated for 1 h with increasing doses of the inhibitor molecules in the presence or absence of 1 nM hCG, as indicated. All compounds were diluted in assay buffer (DMEM/F12, 0.1% BSA; Sigma) containing 4% dimethylsulfoxide (0.5% final concentration in the assay). After 1 h stimulation, the cells were lysed and cAMP was assayed using the Tropix cAMP-screen assay (Applied Biosystems, Foster City, CA), according to the manufacturer’s protocol.
Animals
All animal studies were approved by the Institutional Animal Care and Use Committee. Sprague Dawley CD BR rats (Charles River Laboratories, Wilmington, MA) were housed under the following constant environmental conditions: temperature 22 C ± 2, relative humidity 55 ± 10%, 15–20 air changes per hour, and artificial light with a 12-h circadian cycle (0700–1900 h). For the entire duration of the study, the rats were provided with standard pelleted diet and water ad libitum. In some studies, hypophysectomized rats (Charles River Laboratories) were substituted for intact rats. In this case, rats were 26 d of age at the initiation of the study and were provided 5% sucrose/water during the acclimatization and study periods. Hypophysectomized rats with a body weight greater than 55 g were excluded from the studies. Unmanipulated littermate controls had an average body weight of 87 g (±7 g).
In vivo ovulation induction assay
Immature female rats were weaned at 21–22 d of age and randomly sorted into the experimental groups (n = 6–8/group). The rats were sc injected in the scruff of the neck twice per day (0900 and 1600 h) for 2 d with r-hFSH (606 ng total dose) to induce maturation of multiple ovarian follicles. On the second day and at the same time as the final injection of r-hFSH, all groups received hCG, PDE inhibitor, or a combination of both to induce ovulation of the matured follicles. The vehicle for hCG was PBS, whereas that for the PDE inhibitors was NP3S (5% N-methyl-2-pyrrolidone, 30% polyethylene glycol 400, 25% polyethylene glycol 200, 20% propylene glycol, 20% saline). Preliminary studies using this vehicle demonstrated that at the volumes used, this vehicle neither induced ovulation by itself nor inhibited hCG-induced ovulation.
On the morning after ovulation induction, rats were killed by CO2 asphyxiation. The ovaries, uterine horns, and uterus body were collected and placed in PBS. The oviducts were removed from the ovaries and placed between two glass microscope slides. The oviducts were then examined by light microscopy under phase-contrast conditions and the ova present in the ampullae of the oviducts were counted.
For studies involving hypophysectomized rats, the rats were primed with FSH twice per day for 4 d before ovulation induction. Because the hypophysectomized rats required higher doses of gonadotropins to generate and ovulate follicles, FSH was dosed at 800 ng/d and hCG at 1600 ng (administered concurrently with last FSH injection). Piclamilast was limited to 1 mg/kg due to toxicity observed at higher doses in these animals.
Serum progesterone determination
Intact immature rats were primed with FSH (606 ng total dose) and sc treated with hCG (2860 ng), PDE4 inhibitor (2, 10, and 50 mg/kg), or NP3S vehicle, as described for the ovulation induction assay. Thirty-six hours after treatment, sera were collected and stored at –20 C until assay. Sera were assessed for progesterone levels using the active progesterone enzyme immunoassay (Diagnostic Systems Laboratories, Webster, TX) according to manufacturer’s protocol.
In vitro fertilization
Immature female rats (22 d of age) were superovulated as described above. The method for in vitro fertilization (IVF) of rat gametes was the protocol described by Toyoda and Chang (15) with minor modifications. All chemicals were from Sigma unless specified. M16 medium was supplemented with 25.1 mM NaHCO3, 26 mM Na lactate, 0.5 mM Na pyruvate (16), and 50 IU penicillin/50 µg streptomycin per milliliter (Life Technologies, Rockville, MD). The IVF medium was prepared under sterile conditions using endotoxin-screened water (Life Technologies) and was not filter sterilized due to an unknown toxin contributed by the filter. Supplements were dissolved in the endotoxin-screened water and filter sterilized through a washed syringe filter (Gelman Sciences, Ann Arbor, MI) before being added to the medium. Fresh medium was prepared once per week, the day before the IVF was performed, and was equilibrated overnight at 37 C in a humidified atmosphere of 5% CO2/95% air. Microdrops of IVF medium (100 µl for ova and 500 µl for sperm) were placed in 35 x 10-mm culture dishes (Corning, Corning, NY) that were subsequently flooded with light white mineral oil and equilibrated overnight along with the medium. Also equilibrated were 24-well culture dishes containing IVF medium covered by a thin layer of mineral oil; these were used for collection and dissection of oviducts. Throughout the following procedures, culture dishes containing medium or microdrops were maintained at 37 C on heated stage warmers on the dissecting microscopes.
Adult male rat sperm donors (3–6 months of age) were caged with an adult female rat for 2 wk before collection of sperm (16). One male rat was used for each IVF procedure and was killed by CO2 asphyxiation. Cauda epididymides were removed, rinsed in 37 C IVF medium, and trimmed of adipose tissue. Multiple punctures were made in the organs and drops of sperm were transferred to four microdrops using sterile microdissecting forceps. Sperm were allowed to capacitate for 2–3 h at 37 C in a humidified atmosphere of 5% CO2/95% air. Percent motility of the sperm in each microdrop was determined, as was the concentration of sperm using a hemacytometer. Superovulated female rats were killed by CO2 asphyxiation 18–24 h after ovulation triggering. Oviducts were removed, placed in the medium of the 24-well culture dishes for rinsing, and transferred to fresh wells of medium. The swollen ampulla of each oviduct was torn open with sterile microdissecting forceps, and the clot of ova in their cumulus mass was expressed into the medium. Ovum masses were transferred to microdrops using pipettors fitted with sterile MultiFlex microcapillary pipette tips (VWR, South Plainfield, NJ). Microdrops were inseminated with 25 µl sperm for a final average concentration of 1.3 x 105 sperm/ml.
At 18–20 h after insemination, ova/embryos were rinsed through three microdrops. Using phase-contrast microscopy, the number of dead, unfertilized, and fertilized ova and cleaved embryos were recorded. Ova were considered to be fertilized if a sperm tail was seen inside the plasma membrane, two polar bodies were present, or two pronuclei were visible. Dead cells were identified by fragmentation of the cytoplasm. Cleaved embryos were at the 2- to 4-cell stage.
In vivo fertility assay
Three cohorts of rats (n = 6) were induced to ovulate as described above with the following modifications. Rats (28 d of age) were treated with pregnant mare serum gonadotropin (PMSG) (3 IU) on d 1 and with FSH (700 ng/d) on d 2 and 3 to induce follicular maturation and enable mating behavior. In previous studies, rats injected with FSH only would not exhibit lordosis (data not shown). After ovulation induction, one cohort of rats was killed for oocyte counting as described above, whereas two cohorts were caged singly with adult male rats of proven fertility. Successful mating was determined by the presence of vaginal plugs on the morning after mating, after which the females were removed to separate cages. On d 11 post mating, the second cohort of animals was killed and the number of viable embryo implantation sites was visualized in the uterine horns. The final cohort of rats was allowed to progress to parturition at which time the number of live born pups was determined.
Statistics
Significance differences in ovulation induction were determined by one-way ANOVA using Origin 7.0 (OriginLab Corp., Northampton, MA). Tukey’s honestly significant difference method was used for P value adjustment for multiple comparisons. Statistical significance was set at P < 0.05 for all assays.
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Results |
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). As expected, the cAMP levels were dramatically increased when the nonselective PDE inhibitor 3-isobutyl-1-methylxanthine (IBMX) was added along with the hCG. hCG, either with or without IBMX, had no effect on the cAMP levels in parental or FSH receptor-expressing JC410 cells (not shown). When the JC410 cells were treated with either of the PDE4 inhibitors alone, no response was observed at concentrations up to 10 µM. In contrast, when piclamilast or mesopram were added to the cells with 1 nM hCG, a dose-dependent increase in cAMP was observed, with EC50 values of 23 and 156 nM, respectively.
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). That this is a general phenomenon mediated by selective PDE4 inhibitors is supported by additional studies in which three other selective PDE4 inhibitors, namely WAY-PDA-641 (17), LAS-31025 (18), and CI1044 (19), were also found to induce ovulation when administered to FSH-primed rats (data not shown).
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, 36 h after treatment with either hCG or the PDE4 inhibitor, significantly elevated progesterone levels were detected, indicating that corpora lutea generated after PDE4 inhibitor treatment were fully mature. Correspondingly, corpora lutea were clearly visible on ovaries collected from the hCG and PDE4 inhibitor treated but not on vehicle-treated FSH-primed rats.
Unlike the PDE4 inhibitors piclamilast (IC50 1.5 nM) (21) and mesopram, the PDE4 inhibitor DA-TC-46 (IC50 16 nM) (11) was not able to trigger ovulation at even the highest dose tested. However, in combination with a subeffective dose of hCG, even this inhibitor was capable of inducing more than 25 oocytes/rat at 50 mg/kg (Fig. 3). Similar synergy could be observed between the subeffective dose of hCG and low doses of piclamilast and mesopram (not shown). These results demonstrate synergy between PDE4 inhibition and hCG in triggering ovulation, but treatment with potent PDE4 inhibitors alone is sufficient to mediate this response. This does not exclude the possibility that in hCG-untreated immature rats, there may be a low level of endogenous LH that can be potentiated by inhibition of the regulatory PDE4. In contrast to the ovulatory effects of PDE4 inhibitors, treatment with either the PDE3-selective inhibitor milrinone or the PDE5-selective inhibitor sildenafil citrate had no effect on the number of oocytes that were ovulated when administered either alone or with the subeffective dose of hCG under conditions identical with those in which the PDE4 inhibitors were tested (data not shown).
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). Additionally, treatment with piclamilast in the absence of exogenous hCG induced ovulation in the rats, whereas no ovulation was observed in vehicle-treated rats. These results are consistent with the target cells of PDE4 inhibition being downstream of the pituitary. Coupled with the in vitro and in vivo synergies observed between hCG and PDE4 inhibitors, it is likely that the target for the PDE4 inhibitors are the LH receptor-expressing ovarian cells.
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). The overall fertility and cleavage rates of these oocytes were similar to those that were induced to ovulate with hCG. Because no oocytes were ovulated in control rats that received neither piclamilast nor hCG, all of the collected oocytes were associated with one of these treatments.
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). In contrast, treatment with the PDE4 inhibitor induced a relatively high level of ovulation in all of the rats evaluated. Moreover, the oocytes from the PDE4 inhibitor-treated group were capable of being fertilized in vivo and developing into grossly normal pups at full term. Notably, hCG triggered ovulation in all of the rats in that group, but no viable embryos or live pups were observed after mating.
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Discussion |
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Previous in vivo studies are consistent with a link between PDE4 inhibition and LH signaling. Treatment of mice with rolipram induced premature luteinization of granulosa cells in ovarian follicles (3). Similarly, PDE4D-null mice showed a reduced capacity to ovulate, a small litter size, and a reduced response to superovulation due to the capture of oocytes within luteinized follicles (24). These published results and those of the present study are indicative of the critical timing of PDE4 inhibition in determining the nature of the ovarian response. Chronic PDE4 inhibition, particularly during follicular maturation, may be more likely to mimic a premature LH-like response, whereas inhibition of PDE4 after FSH-induced follicular maturation may mimic the normal LH surge.
PDE4 enzymes are encoded by four homologous genes, PDE4A to PDE4D, each of which may express numerous isoforms (7). Genetic deletion of PDE4D results in a clear reproductive phenotype, and both PDE4B and PDE4D have been found to be expressed in ovarian follicles by in situ hybridization, with PDE4B localized to the thecal and interstitial cells and PDE4D to the mural (but not cumulus) granulosa cells of antral follicles (8). Because LHR expression has been demonstrated in both of these cell types (25), it is unclear whether inhibition of PDE4B, PDE4D, or both is necessary for triggering ovulation. Rolipram, the inhibitor reported to induce premature luteinization in mice, has only a 2-fold selectivity for PDE4B vs. PDE4D (26). Similarly, piclamilast, the most potent inducer of ovulation tested in our model, has little selectivity for the individual PDE4 gene products (27), although this inhibitor is highly selective for the PDE4 family members (28). Exact identification of the PDE4 subtype(s) critical for ovulation must await the identification of subtype-selective inhibitors or the availability of mice genetically deficient in the other PDE4 genes. In addition to the expression of PDE4 in follicular cells, PDE3 isoforms are also expressed in the ovarian follicle (29). As seen in PDE4 knockout mice, mice lacking PDE3 display fertility deficiencies. However, because expression of PDE3 appears to be localized in the oocyte, which lacks gonadotropin receptor expression, it should not be unexpected that an inhibitor of PDE3 was incapable of substituting for or potentiating gonadotropin activity. It should be noted, however, that although infertile, the PDE3 knockout mice were fully capable of ovulating oocytes under normal gonadotropin stimulation. Similarly, inhibition of PDE5, a cGMP-specific phosphodiesterase found in brain, vascular smooth muscle cells, and other tissues (30), also did not induce ovulation under the current experimental conditions. Although other non-type 4 phosphodiesterases have been reported in granulosa cells (3), no evidence that PDE5 is expressed in ovarian-specific tissues has been reported.
Whereas in vitro and in vivo evidence supports a direct role for PDE4 in regulating CG/LH-mediated granulosa cell function, an indirect role for PDE4 in triggering ovulation cannot be ruled out because the PDE4 selective inhibitor rolipram can increase secretion of GnRH from the GT1 neural cell line in vitro (31), and intracerebroventricular administration of the PDE4 inhibitor denbufylline into adult male rats induces the release of LH (32). In the latter study, release of LH may have been mediated by cross-reactivity of the inhibitor with a non-PDE4 target because intracerebroventricular administration of rolipram did not induce LH release. To determine whether the ovulation that was induced with PDE4 inhibitors in the present study was mediated through the release of LH from the pituitary, the use of hypophysectomized rats was employed. Whereas the overall sensitivity of these rats to gonadotropins was less than that observed in intact rats, the fact that the PDE4 inhibitor piclamilast was capable of inducing ovulation supports a direct action of the inhibitors on the ovarian cells themselves. Although PDE4 inhibitors strongly synergize with subeffective levels of hCG in inducing cAMP in rat (not shown) and porcine granulosa cells in vitro, it should be noted that we were unable to detect a change in ovarian cAMP levels after treatment of FSH-primed rats in vivo with the PDE4 inhibitors at ovulatory doses, possibly supporting an indirect role of the inhibitors in ovarian function. However, because we also could not detect ovarian cAMP changes after hCG treatment in vivo, it is possible that a biologically sufficient cAMP response took place but was undetected, either due to the transient nature of the response or masking by the FSH-induced cAMP. Recently Park et al. (33) provided evidence that LH signaling through the LH receptor induces the downstream release of epithelial growth factor-like growth factors which, in turn, mediate many of the LH-associated follicular responses such as cumulus expansion and oocyte maturation. This paracrine mechanism cascades the LH signal throughout the ovarian follicle. Whereas results presented in our study are consistent with a direct role for PDE4 in regulating LH receptor activity, it remains possible that the inhibitors are in fact involved in the regulation of the signaling of some or all of these downstream peptide mediators.
In the clinic, hCG is typically administered sc to induce ovulation as part of clinical ovulation induction programs in the treatment of infertility. The identification of orally active small molecules that can substitute for gonadotropin activity may make such injectable therapeutics unnecessary. From the present results, we suggest that inhibitors of PDE4 may be suitable for the induction of ovulation as part of an assisted reproduction technology protocol. Aside from practical and economical considerations, such a treatment regimen could potentially reduce the risk of ovarian hyperstimulation syndrome, a rare but serious complication of a clinical ovarian stimulation regimen. The development of this syndrome has been linked to the prolonged activity of hCG in treated women (34). Therefore, the use of small molecules that have relatively rapid clearance rates and/or affect only one aspect of the hCG/LH signal transduction pathway may be advantageous. As demonstrated in the present fertility studies, oocytes recovered after PDE4 inhibition were competent to be fertilized in vitro, and in vivo fertility, as measured by the development of viable embryos and normal full-term pups, was markedly greater than that after hCG-induced ovulation. Of potential relevance, Mattheij et al. (35) reported that administration of 20 IU hCG to rats during late diestrus or proestus before mating results in embryonic mortality at some point after d 3 of pregnancy but before implantation. The authors speculate that the hCG disrupted steroidogenesis, which may have then interfered with implantation. However, the LHR has been demonstrated to be expressed in the oviduct and stromal cells of the uterine endometrium of mice and rats (36), so a direct role for hCG on extragonadal tissue in mediating this mortality cannot be ruled out. Based on our results, the hCG-mediated embryo toxicity may have been circumvented by the use of PDE4 inhibitors in place of hCG.
In summary, the results presented herein demonstrate the utility of PDE4-selective inhibitors in triggering ovulation. This notion supports the concept that targeting components of gonadotropin signal transduction pathways with small molecules may provide an alternative to the use of injectable protein therapeutics in the treatment of infertility.
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Footnotes |
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Abbreviations: DC-TA-46, 7-Benzylamino-6-chloro-2-piperazino-4-pyrrolidino-pteridine; hCG, human chorionic gonadotropin; IBMX, 3-isobutyl-1-methylxanthine; IVF, in vitro fertilization; LHR, LH receptor; PDE, phosphodiesterase; PMSG, pregnant mare serum gonadotropin; r-h, recombinant human.
Received May 4, 2004.
Accepted for publication September 9, 2004.
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generation from human monocytes by interacting with a ‘low-affinity’ phosphodiesterase conformer. Br J Pharmacol 118:649–658[Medline]
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