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Molecular Endocrinology, doi:10.1210/me.2002-0435
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Molecular Endocrinology 17 (6): 1117-1130
Copyright © 2003 by The Endocrine Society

Phosphodiesterase Regulation Is Critical for the Differentiation and Pattern of Gene Expression in Granulosa Cells of the Ovarian Follicle

Jy-Young Park1, Francois Richard1, Sang-Young Chun, Jeong-Hoh Park, Evelyn Law, Kathleen Horner, S-L Catherine Jin and Marco Conti

Division of Reproductive Biology (J.-Y.P., F.R., E.L., K.H., S.-L.C.J., M.C.), Department of Obstetrics and Gynecology, Stanford University, Stanford, California 94305; and Hormone Research Center (S.-Y.C., J.-H.P.), Chonnam National University, Kwangju 500-757, Republic of Korea

Address all correspondence and requests for reprints to: Marco Conti, Division of Reproductive Biology, Department of Obstetrics and Gynecology, Stanford University School of Medicine, 300 Pasteur Drive, Stanford, California 94305-5317. E-mail: marco.conti{at}stanford.edu.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Feedback regulations are integral components of the cAMP signaling required for most cellular processes, including gene expression and cell differentiation. Here, we provide evidence that one of these feedback regulations involving the cyclic nucleotide phosphodiesterase PDE4D plays a critical role in cAMP signaling during the differentiation of granulosa cells of the ovarian follicle. Gonadotropins induce PDE4D mRNA and increase the cAMP hydrolyzing activity in granulosa cells, demonstrating that a feedback regulation of cAMP is operating in granulosa cells in vivo. Inactivation of the PDE4D by homologous recombination is associated with an altered pattern of cAMP accumulation induced by the gonadotropin LH/human chorionic gonadotropin (hCG), impaired female fertility, and a markedly decreased ovulation rate. In spite of a disruption of the cAMP response, LH/hCG induced P450 side chain cleavage expression and steroidogenesis in a manner similar to wild-type controls. Morphological examination of the ovary of PDE4D-/- mice indicated luteinization of antral follicles with entrapped oocytes. Consistent with the morphological finding of unruptured follicles, LH/hCG induction of genes involved in ovulation, including cyclooxygenase-2, progesterone receptor, and the downstream genes, is markedly decreased in the PDE4D-/- ovaries. These data demonstrate that PDE4D regulation plays a critical role in gonadotropin mechanism of action and suggest that the intensity and duration of the cAMP signal defines the pattern of gene expression during the differentiation of granulosa cells.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
ALTHOUGH MUCH IS known about the machinery involved in the generation of the second messenger cAMP, it is still uncertain how different and often opposing effects may be produced in a cell in response to the activation of this signaling pathway (1). Drawing from what is known about Ca2+ signaling (2), it can be hypothesized that information is not exclusively conveyed by changes in the concentration of the second messenger cAMP; rather, spatial and temporal dimensions of the cAMP transient are critical components of the signals required for different cell functions and different patterns of gene expression. Indeed, in T cells, changes in the frequency and duration of the Ca2+ responses produce distinct patterns of gene expression, cytokine production, and ultimately different T cell activations (2).

cAMP is generated after the interaction of ligands with a receptor coupled to a transducer stimulatory guanine nucleotide binding protein (Gs) protein (3). The occupied receptor promotes the exchange of GTP in the transducer, thus generating an activated {alpha}-subunit, which in turn activates the effector adenylyl cyclase. The activation of this membrane signal transduction machinery is transient because several mechanisms are activated to terminate the stimulation and to cause a return of the cell to a resting state. These include the phosphorylation of the receptor by different kinases and recruitment of ß-arrestins, or inactivation of Gs via hydrolysis of GTP at a rate controlled by the regulator of G protein signaling (RGS) protein (4). The termination of the cAMP stimulus is critical for correct signaling, as several knockout (KO) models indicate that inactivation of desensitization mechanisms produces major disruptions in cell function (5). Downstream of receptor/G protein/effector coupling, the regulation of phosphodiesterases (PDEs) is an additional mechanism of termination of the stimulus/desensitization distal to the generation of cAMP (6). Several studies indicate that PDEs are involved in feedback mechanisms that control cAMP levels (6, 7).

Of the 11 known families of PDEs (8), PDE4s are the target of a cAMP feedback regulation mediated either by rapid phosphorylation or by changes in PDE expression. These genes are the mammalian orthologs of the Drosophila dunce gene involved in learning, memory, and female fertility of the fly (6). In thyroid cells (9), Sertoli cells (10), T cells (11), monocytes (12), and neurons (13), an increase in cAMP is followed by increased transcription of PDE4 genes. In most cases, transcription from intronic promoters sensitive to cAMP direct the accumulation of mRNAs coding for the short PDE4 variants. On the basis of in vitro studies, it has been proposed that this long-term regulation is involved in desensitization in Sertoli and thyroid cells (14, 15).

In the ovary, growth and differentiation of the follicle requires the concerted regulation by gonadotropins as well as local regulators (16, 17). Both FSH and LH act through GPCRs that belong to the subfamily of leucine repeat receptors (18). Although coupling to other G proteins has been reported, it is accepted that gonadotropin receptors signal through activation of Gs and adenylyl cyclases. The ensuing increase in cAMP activates a complex pattern of gene expression causing rapid cell replication and steroidogenesis. After LH stimulation, an increase in cAMP produces profound changes in expression of those genes required for switching the pattern of steroidogenic hormone production (19, 20), withdrawal from the cell cycle (21, 22), and altering the structure of the follicle wall to allow ovulation (22, 23). How these changes are produced by a seemingly simple cAMP signal is unclear. Coupling of the gonadotropin receptors to other signaling pathways, or changes in the properties of the cAMP signal itself, may be the basis of this complex differentiative program.

Here, we provide evidence that PDE4D is one of the genes regulated by gonadotropin stimulation of granulosa cells. Inactivation of this PDE gene by homologous recombination causes a disruption of the cAMP signal by altering its intensity, duration, and probably its spatial propagation. As a result, the pattern of gene expression required for differentiation of granulosa cells is subverted, with genes involved in ovulation not expressed, whereas genes involved in luteinization are induced normally or perhaps prematurely. Furthermore, an altered cAMP signaling due to ablation of the PDE feedback and the consequent disruption of gene expression produces a major impairment in fertility.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
PDE4D Is a Gonadotropin-Inducible Gene
Previous studies have indicated that an increase in PDE activity follows gonadotropin stimulation in vitro and that PDE4D mRNA is present in granulosa cells as determined by in situ hybridization (24, 25). As a first step to investigate the function of PDE4D in the follicle, gonadotropin regulation of PDE4D mRNA expression in vivo was investigated using ribonuclease (RNase) protection with probes hybridizing to all five PDE4D transcripts or probes that distinguish between the short and long PDE4D forms (26). In vivo pregnant mare serum gonadotropin (PMSG) treatment induced an increase in PDE4D mRNA that reached a maximum at 6–12 h and remained elevated even after 48 h (Fig. 1AGo). Using probes that distinguish between the different splicing variants of PDE4D, it was determined that PMSG treatment induces the accumulation of mRNA coding for the short forms PDE4D1 and PDE4D2, and to a lesser extent those of the long forms (Fig. 1Go, B and E). A similar pattern of PDE4D induction was observed in more mature granulosa cells stimulated by human chorionic gonadotropin (hCG; Fig. 1CGo). In the latter case, however, the mRNA induction reached a maximum in 1 h and decreased thereafter. Again, mRNAs coding for the short variants were the transcripts stimulated by hCG (Fig. 1DGo). Conversely, the mRNAs coding for the long forms was minimally increased after 1 h (Fig. 1Go, D and F).



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  		Figure 1. Hormonal Regulation of PDE4D mRNA in Rat Ovary

A group of immature rats were injected with PMSG; animals were killed at different times after the injection and ovaries removed. An additional group of animals received an hCG injection 48 h after PMSG. Total RNA (20–30 µg) from PMSG-treated (A and B) and PMSG/hCG-treated (C and D) ovaries was analyzed by RPA using either a probe common for all PDE4D (A and C) or two probes that distinguish between the long and short splicing variants of PDE4D (B and D). Intensity of the protected bands was quantitated by densitometry and the GAPDH was used for normalization of the data (E and F). Data reported are the mean ± SEM of two or three separate experiments.

 
This increase in PDE4D mRNA steady state is followed by an increase in total PDE activity measured in ovarian extracts (Fig. 2Go). When the PDE4-specific inhibitor rolipram was used to distinguish between the PDE4 and other PDE activities, the hCG stimulation was no longer evident (Fig. 2Go), indicating that the activation is restricted to PDE4 forms and that rolipram-insensitive PDEs are not affected by the gonadotropin stimulation. Furthermore, Western blot analysis with a PDE4D-selective antibody showed that a large increase in PDE4D1/D2 follows hCG stimulation, whereas the expression of the long forms is not increased (Fig. 2Go, inset).



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  		Figure 2. Gonadotropin Stimulation of PDE4 Activity and Protein Accumulation in the Ovary in Vivo

Immature mice were primed with PMSG. After 48 h, one group received an hCG injection. After PMSG priming for 48 h, mice were killed (hCG; 0 time) or injected with hCG and killed after 2 h. Ovaries were homogenized under conditions detailed in Materials and Methods. Aliquots of homogenates were assayed for total PDE activity and rolipram-insensitive PDE activity in the presence of 10 µM rolipram. Rolipram-sensitive activity was obtained by subtracting the rolipram-insensitive PDE activity from total PDE activity. The data are the mean ± SEM of three separate experiments. Inset, Whole ovary extracts were subjected to immunoprecipitation with a pan-PDE4 antibody (K116) and Western blot performed with a PDE4D-selective antibody. A representative experiment of the two performed is included.

 
Collectively, these data demonstrate that PDE4D is a gonadotropin-regulated gene and that activation of transcription of this gene is followed by increased PDE4D protein accumulation and activity. Thus, the feedback regulation involving cAMP/PDE4D is functional in granulosa cells in vivo as it has been shown to be in other endocrine cells (6). At present, we could not determine the impact of long form phosphorylation on the increase in PDE activity observed after hCG.

Mice Deficient in PDE4D Have a Defect in Ovulation and Follicle Maturation
As previously reported (27), female mice deficient in PDE4D have a reduced litter size. In further studies, it was observed that, out of five adult females followed over a period of 6 months, only one consistently produced litters (five of six matings), three produced one litter in five matings, and one never exhibited signs of pregnancy (data not shown). The decreased fertility is associated with a decrease in ovulation rate and an increase in oocyte degeneration (27). An impaired ovulation is mostly evident in superovulation experiments using immature mice. PMSG treatment followed by hCG stimulation causes massive ovulation after 18 h in wild-type littermates (Fig. 3Go). Conversely, only few, often denuded, oocytes were recovered in the ampulla of the PDE4D-/- mice (Fig. 3Go). The ovulation rate of heterozygous females was only marginally decreased when compared with wild-type littermates, confirming the normal fertility of these mice (Fig. 3Go).



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  		Figure 3. Reduced Ovulation Rate in PDE4D-/- Mice

Immature mice were treated with PMSG and hCG for superovulation as described in Materials and Methods. Ovulated oocytes collected from the ampulla of the oviducts were counted and reported as the number of oocytes ovulated/mouse. The ovulation rate of PDE4D-/- mice is significantly different from that of PDE4D+/+ mice (P < 0.001 determined by ANOVA). Number of mice used was 19, 26, and 20 for the three genotypes.

 
To investigate the cause of this large decrease in ovulation efficiency, the development of the ovarian follicle was followed in mice deficient in PDE4D. Immature ovaries from 10-d-old mice were grossly normal, suggesting that PDE4D ablation does not affect early follicle development (data not shown). However, superovulation experiments in immature (20–30 d old) mice uncovered a disruption in follicle final maturation. Although the number of large antral follicles was comparable in wild-type and PDE4D-/- littermates, and some normal preovulatory follicles were present 48 h after the PMSG stimulation (Fig. 4Go), numerous structures resembling luteinizing follicles were evident in the ovaries from the PDE4D-/- mice (Fig. 4Go). Upon further inspection, most of these follicles had entrapped oocytes that had undergone germinal vesicle breakdown and had lost contacts with cumulus cells. Granulosa cells had signs of hypertrophy, with elongated nuclei, and marked eosinophilia and increased volume of the cytoplasm, features typical of luteinizing granulosa cells (Fig. 4DGo). A count of the luteinizing follicles in serial sections showed major differences between the three genotypes (luteinized follicles with entrapped oocytes/ovary: wild-type, <1; PDE4D+/-, 7.6 ± 1.8; PDE4D-/-, 18.1 ± 2.3; n = 4).



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  		Figure 4. Histological Appearance of PDE4D+/- and PDE4D-/- Ovaries

Ovaries from immature 20- to 25-d-old mice treated with PMSG for 2 d were fixed, sectioned, and stained with hematoxylin/eosin. Normal large antral follicles and follicles with signs of luteinization and/or degenerating oocytes are indicated by the black and white arrows, respectively, in PDE4D+/- and PDE4D-/- mice (A and B). High magnification (x400) of a representative follicle from the PDE4D+/- and PDE4D-/- ovaries is reported in C and D, respectively (bar, 50 µm).

 
In some instances, oocytes were recovered in the fallopian tubes of PMSG-treated PDE4D-/- mice, suggesting aberrant rupture of the follicles. These oocytes were denuded and with signs of degeneration.

Altered cAMP Responses in Vivo and in Vitro in Granulosa Cells of the PDE4D-/- Mice
cAMP content was measured in the ovary of wild-type and PDE4D null mice after in vivo stimulation with an ovulatory dose of hCG (Fig. 5AGo). cAMP levels increased rapidly 30 min after hCG treatment in wild-type ovaries and decreased thereafter in coincidence with the PDE4D induction. A decrease in resting cAMP levels was observed in the PDE4D null ovaries 48 h after PMSG (0 time). More importantly, the time course of cAMP accumulation in response to hCG was altered, with cAMP reaching a maximum at 1 h and remaining at the same level thereafter, suggesting that the absence of the PDE4D prevents a rapid removal of the second messenger. A consistent difference in maximal response was also observed (Fig. 5AGo). The effect of stimulation with gonadotropins was further studied in vitro in a more controlled population of granulosa cells derived from preovulatory follicles (Fig. 5BGo). In these in vitro cultures, gonadotropin stimulation was again impaired in the PDE4D-/- granulosa cells. Conversely, forskolin, which bypasses the receptor/G protein activation of cyclase, produced a normal, or in some instances elevated, cAMP accumulation (Fig. 5BGo). These data demonstrate that PDE4D-/- granulosa cells display a decreased response to gonadotropin and that the lesion is at the level of receptor/G protein coupling because the forskolin response is normal. The levels of the LHR mRNA measured by Northern blot were comparable in wild-type and PDE4D-/- mice (data not shown). On the basis of these findings, we surmised that the removal of the PDE4D feedback in granulosa cells is associated with an altered pattern of cAMP accumulation and a compensatory uncoupling of the receptors from G protein/cyclase activation.



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  		Figure 5. cAMP Responses to Gonadotropin Stimulation of Mouse Ovaries in Vivo (A) and Granulosa Cells in Vitro (B)

A, PMSG-primed PDE4D+/+ and PDE4D-/- mice were injected with an ovulatory dose of hCG and ovaries were collected at different times after injection. cAMP accumulation in the ovary was measured by RIA as described in Materials and Methods. cAMP accumulation in the PDE4D+/+ ovary was significantly different from that of PDE4D-/- ovaries at 0 (n = 10–13), 0.5 (n = 6–9), and 3 (n = 5) h after hCG treatment (***, P < 0.001; * P < 0.05). B, Granulosa cells were retrieved from preovulatory follicles of ovaries of PMSG-primed mice. After washing, granulosa cells were incubated in serum-free conditions in the presence of 250 ng/ml FSH, 100 ng/ml hCG, or 10 µM forskolin for 1 h. Incubation was terminated by addition of TCA, nucleotides were extracted, and cAMP concentration was measured by RIA. The mean and SEM of three to six observations for each group is reported.

 
Gonadotropin Activation of the Steroidogenic Program Is Not Affected in the PDE4D Null Ovary
The above data suggest that ablation of the PDE4D feedback in granulosa cells produces an aberrant gonadotropin-dependent cAMP signal. Although cAMP accumulation is still present, the intensity and duration of this stimulus is altered. Therefore, this genetic model was used to investigate whether this aberrant cAMP signal impacts the pattern of gene expression in mature follicles.

To determine whether the transition of granulosa cells to luteal cells is disrupted in the PDE4D-null mice, the induction of the P450 side chain cleavage (P450scc) gene was investigated. In spite of a decrease in the cAMP signal, in vivo hCG treatment produced a robust stimulation of P450scc mRNA accumulation in the PDE4D-/- ovaries (Fig. 6Go). In addition, a moderate but consistent increase in the initial P450scc mRNA level was observed (Fig. 6BGo). A normal stimulation of steroidogenesis by LH/CG was confirmed by measuring progesterone accumulation by RIA (Fig. 7Go). The expression of P450scc in follicles at different stages of maturation was monitored by in situ hybridization (Fig. 8Go). In wild-type ovaries from mice treated with PMSG for 48 h, expression of P450scc was confined to interstitial/theca cells. In rare instances, some signal was evident in the basal layers of mural granulosa cells. In contrast, P450scc mRNA was expressed in granulosa cells in numerous follicles of the PDE4D-/- ovaries (Fig. 7Go). This finding is consistent with the partially luteinized phenotype of granulosa cells.



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  		Figure 6. P450scc Gene Expression in PDE4D+/- and PDE4D-/- Ovaries

PMSG-primed immature mice were injected with hCG and ovaries collected at different times after stimulation. Total RNA (20 µg) was analyzed by Northern blot using a cDNA probe for mouse P450scc or GAPDH (A). The abundance of the P450scc transcripts was quantitated by densitometry, and data were expressed as the ratio of the P450scc/GAPDH transcript in each sample (B).

 


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  		Figure 7. Gonadotropin Stimulation of Serum Progesterone in PDE4D+/+, PDE4D+/-, and PDE4D-/- Mice

Immature mice were primed with PMSG only or 24 h after hCG injection. Progesterone levels were measured with serum by RIA. n = between 8 and 16.

 


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  		Figure 8. In Situ Expression of P450scc mRNA in Ovaries from Immature PMSG-Treated PDE4D+/+, and PDE4D-/- Mice

Immature mice were treated with PMSG for 48 h. At the end of the treatment, ovaries were excised and processed for in situ hybridization as detailed in Materials and Methods; PDE4D+/+ (A and C) PDE4D-/- (B and D). A representative experiment of the four performed is reported.

 
In the same vein, the induction of genes involved in withdrawal from the cell cycle was apparently not affected in the PDE4D-/- granulosa cells. After hCG stimulation, an increase in mRNA coding for the CDK inhibitor p27kip1 was more evident in the PDE4D-/- granulosa cells than in wild-type controls (data not shown). Again, p27kip1 mRNA levels 48 h after PMSG were slightly increased, when compared with wild-type controls.

On the basis of these findings, we concluded that the pattern of gene expression involved in luteal cell differentiation is activated normally by LH/hCG in PDE4D-/- granulosa cells. These findings are consistent with the morphological observation of the luteinization of granulosa cells in the PDE4D-/- mice.

The Pattern of Gene Expression Involved in Ovulation Is Disrupted in the PDE4D-/- Follicle
At the time of luteinization, the LH surge also activates a program of gene expression required for oocyte maturation and ovulation (28, 29). The induction of the progesterone receptor is a key step in this program as PR null follicles do not ovulate (22). In addition, it is generally accepted that progesterone and its receptor are required for the expression of downstream genes, including proteases and locally released peptides required for follicular rupture (30, 31). To investigate whether PR gene expression is activated in the PDE4D null ovary, the mRNA levels for this transcription factor were followed at different times after in vivo stimulation with hCG. In agreement with previous data, wild-type or heterozygous ovaries expressed PR mRNA transiently with a maximum reached 1–3 h after hCG stimulation (Fig. 9Go). Conversely, the PR mRNA was not induced, or was induced at greatly reduced levels, in the PDE4D null ovaries (Fig. 9Go). Consistent with this finding, the expression of two genes thought to be downstream of PR, cathepsin L (Fig. 10Go), and PACAP (Fig. 11Go), was also markedly decreased in ovaries deficient in PDE4D.



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  		Figure 9. PR Gene Expression in PDE4D+/- and PDE4D-/- Ovaries

Ovaries from immature mice were collected as described in Fig. 6Go. Total RNA (2 µg) from a set of ovaries from two mice was used for semiquantitative RT-PCR as described in Materials and Methods using specific PR primers (A). GAPDH was used for normalization and quantitation of the data (B). Data are reported as the mean ± SEM of the ratio of PR/GAPDH density. n = 3 independent experiments.

 


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  		Figure 10. Cathepsin-L Gene Expression in PDE4D+/- and PDE4D-/- Ovaries

Total RNA (20 µg) from ovaries of mice treated with hCG for 12 h were used for Northern blotting analysis (A). The intensity of the bands were quantitated by densitometry, and the data were normalized with ß-actin (B; n = 6 mice/group).

 


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  		Figure 11. PACAP Gene Expression in PDE4D+/- and PDE4D-/- Ovaries

Ovaries from immature mice were collected as described in Fig. 6Go. Total RNA (2 µg) from ovaries of two mice was used for semiquantitative RT-PCR as described in Materials and Methods using specific mouse PACAP primers (A). GAPDH was used for normalization and quantitation of data (B). Data are reported as the mean ± SEM of the ratio of PR/GAPDH density (n = 3 independent experiments).

 
An additional branch of the gene expression pathway involved in ovulation is the activation of prostanoid synthesis after induction of cyclooxygenase-2 (COX-2; Refs. 32 and 33). The induction of this mRNA was severely blunted in the PDE4D ovaries (Fig. 12Go), demonstrating that the inactivation of PDE4D affects the entire program of gene expression involved in ovulation. In situ hybridization analysis of the COX-2 mRNA distribution 3 h after hCG stimulation confirmed that the majority of the follicles in the PDE4D-/- ovary did not express this mRNA. Expression was present in the few follicles that had not luteinized (data not shown).



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  		Figure 12. COX-2 Gene Expression in PDE4D+/- and PDE4D-/- Ovaries

Ovaries from immature mice were collected as described in Fig. 6Go. Total RNA (2 µg) from a set of two ovaries was used for semiquantitative RT-PCR as described in Materials and Methods using specific COX-2 primers (A). GAPDH was used for normalization and quantitation of the data (B). Data are reported as the mean ± SEM of the ratio of PR/GAPDH density (n = 3 independent experiments).

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
MATERIALS AND METHODS
 REFERENCES
 
The differentiation of the ovarian follicle involves highly coordinated programs of gene expression in granulosa cells. The initial expression of genes required for a rapid expansion of the granulosa cell pool is switched off by a coordinated extinction/activation of genes that direct the withdrawal from the cell cycle, followed by expression of genes involved in luteinization and ovulation. Our findings demonstrate that the PDE4D induction is critical for the correct execution of the above programs. Inactivation of this cAMP/PDE4D regulatory feedback disrupts the timing and coordination of the pattern of gene expression and ovulation, ultimately leading to impaired fertility. More importantly, our data show that a disruption of cAMP signaling by inactivation of PDE4D has distinct and opposing effects on two limbs of the differentiative programs activated by LH/hCG in granulosa cells. Thus, PDE4D plays a critical role in determining the specificity of cAMP signaling and the distal outcomes.

Our data demonstrate that PDE4D mRNA is induced in granulosa cells during follicle development in vivo by both FSH and LH/hCG. On the basis of our previous work on the structure and promoter regulation of the PDE4D gene, it is likely that gonadotropins regulate the transcription from an intronic promoter and the accumulation of mRNAs coding for the short PDE4 variants (10, 34). These variants are catalytically active, as documented by the increase in PDE protein and activity measured in the ovary. Numerous studies using cell culture models indicate that PDE4D induction constitutes a feedback regulation of cAMP levels (35). Therefore, this cAMP/PDE feedback is operating in granulosa cells in vivo.

The ablation of PDE4D produces two major effects on cAMP signaling in follicular cells. Unlike the wild-type ovary where the cAMP increase is transient, cAMP levels in the PDE4D null ovary increase to a new steady state but then remain constant. This finding is consistent with the idea that the PDE4D feedback is required to generate a transient response and that the induction of PDE4D1/2 causes a return of cAMP to basal levels in granulosa cells 1–2 h after hCG. In contrast with what one would expect from ablation of a PDE, cAMP responses to LH were also reduced in the PDE4D null granulosa cells both in vitro and in vivo. Because the response to forskolin is normal or increased, it appears that the decreased gonadotropin stimulation of cAMP is due to a lesion upstream of the adenylyl cyclase itself, most likely an impaired coupling between the LH receptor and the Gs protein. We have concluded that this reduced response reflects an adaptive mechanism to the loss of PDE4D causing LH receptor desensitization/down-regulation. This hypothesis is based on the observation that LH receptor mRNA was not significantly altered in the PDE4D-/- ovary, and on the finding that up-regulation of other PDEs does not occur after ablation of PDE4D. The concept that PDE4D induction is a critical component of desensitization recently has been underscored by the finding that this cAMP degrading enzyme is recruited to the phosphorylated ß-adrenergic receptor via binding to ß-arrestin (36). It is then quite plausible that removal of this desensitization mechanism is associated with the activation of compensatory mechanisms, such as a protein kinase A-mediated phosphorylation of the receptor or other mechanisms of uncoupling and internalization.

Regardless of the molecular mechanisms underlying the altered response to hCG/LH, inactivation of the PDE4D feedback causes an overall disruption in the pattern of cAMP accumulation in granulosa cells. It should be emphasized that the cAMP signal is still present in response to hCG and that only its intensity and duration is affected both in vitro and in vivo. With these premises, the PDE4D KO model provides a unique opportunity to investigate the effect on distal responses of an altered, but not completely severed, cAMP signaling pathway.

A morphological analysis of the follicle, as well as the analysis of the pattern of granulosa cell gene expression, demonstrated that an altered cAMP signaling compromises the program of ovulation but has minor effects on luteinization. That luteinization occurs, perhaps prematurely, is documented by the morphology of the follicle as well as by the normal induction of P450scc, normal progesterone production, and regulation of the G1 cyclin/CDK inhibitor p27Kip1. Thus, key genes involved in the changes in steroidogenesis and withdrawal from the cell cycle are induced in spite of an altered cAMP signal. Conversely, the program controlling follicle rupture and ovulation is profoundly affected. This conclusion is based on the large decrease in the induction of both the PR mRNA and the two genes thought to be distal to PR, on the impaired expression of COX-2, and on the large reduction in ovulation rate of the PDE4D-/- mice. These findings, therefore, demonstrate that two branches of the program of gene expression activated during ovulation and luteinization are dissociated in the PDE4D null mice. Furthermore, the overlapping phenotypes of impaired ovulation in the PDE4D, PR, and COX-2 KO mice is consistent with our observation that the latter two genes are poorly induced after inactivation of PDE4D. In light of these findings, we can conclude that PDE4D is epistatic to the PR and COX-2 genes in the cascade of gene expression regulated by LH.

The transcription of most genes activated by LH in granulosa cells, including P450scc, COX-2, and PR, is mediated by an increase in cAMP and can be reproduced by stimulating granulosa cells with forskolin or cAMP analogs; moreover, functional CRE cis-acting elements are present in the proximal promoter of two of these genes (37, 38, 39, 40, 41). Therefore, the dissociation of the two patterns of gene expression in the PDE4D KO mice cannot be explained simply by the absence of cAMP cis-acting elements in the promoters of the genes that are still induced. Rather, we propose that the intensity and duration of the cAMP signal determines the pattern of gene expression in granulosa cells, and the combination of cis-acting elements in the different promoters defines the sensitivity to the cAMP stimulus. Whereas the expression of PR and COX-2 requires a robust and transient cAMP signal, P450scc transcription is activated with minimal but prolonged changes in cAMP.

Although not addressed here, the spatial dimension of the cAMP signal may play an additional important role in the differential regulation of transcription. That cAMP signal compartmentalization is present in the cell is indicated by studies in heart, where cAMP increases in discrete regions of the cell, and little diffusion from these microdomains can be detected (42, 43). More importantly, several findings indicate that PDEs play an important role in preventing diffusion of cAMP and defining the signaling in a membrane cAMP microdomain (44). In addition, protein kinase As and PDE4Ds are anchored to subcellular domains by similar A kinase anchor proteins (45, 46). Thus, it is possible that the absence of PDE4D in granulosa cells alters the spatial propagation of the cAMP signal and that this in turn affects transcription in a selective manner.

Several observations support the view that the cAMP signal in granulosa cells branches to activate other pathways, including the activation of pathways that involve protein kinase C-, MAPK-, or protein kinase B/serum and glucocorticoid-inducible kinase-mediated phosphorylations (47). In vitro activation of protein kinase C by GnRH or phorbol 12-myristate 13-acetate can support luteinization (48). It is thus possible that the PDE4D ablation disrupts the distributing of the cAMP signal to these distal pathways. Transcription that requires the coordinated stimulation of cAMP and other signaling pathways still occurs in the PDE4D KO mice. Conversely, transcription of genes relying primarily on cAMP signaling is impaired. The mouse model that we have generated will therefore be a powerful tool to dissect these pathways downstream of the LH receptors.

Dysfunction of ovulation is a primary cause of female infertility and the syndrome of luteinized unruptured follicles is a relatively common phenomenon in infertile women (49). Thus, the phenotype of the PDE4D KO mouse suggests that inactive alleles of the PDE4D gene may be a cause of decreased ovulation efficiency and infertility in humans. Although the PDE4D locus has not been linked previously to cases of infertility, this is a possibility that should be explored further.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
REFERENCES
 
Animals
PDE4D-/- mice were generated as previously described (27). Genotype analysis was performed by PCR or Southern blotting as described (27).

Mice were housed in a room with controlled temperature and a photoperiod of 12 h dark, 12 h light. Food and water were provided ad libitum. Immature female mice, ranging in age from 20–30 d (body weight 10–12 g), received an ip injection of 5 U PMSG (Calbiochem, La Jolla, CA). Immature (26 d old; body weight, 60–65 g) female Sprague Dawley rats (Daehan Laboratories, Chungbuk, Korea) were injected sc with 10 IU PMSG. After 46–48 h, animals were killed by cervical dislocation, and the ovaries were removed. Some animals were injected with 5 U hCG (Goldline Labs, Fort Lauderdale, FL) to induce ovulation, and ovaries were obtained at different time intervals for the experiments.

Granulosa Cell Isolation and Culture
Granulosa cells of preovulatory follicles were collected from PMSG-treated ovaries of immature mice. Granulosa cells were isolated by follicle puncture using 23-gauge needles under a micromanipulator and washed twice in fresh HEPES-buffered McCoy’s 5A culture media. Cells were counted using trypan blue and incubated at a density of 1 x 106 cells/60-mm dish in 1 ml of culture media with antibiotics and 0.1% BSA (wt/vol, fraction V; Sigma, St. Louis, MO). Hormones were added and cells were incubated at 37 C in a humidified 95% air/5% CO2 incubator. After incubation, cells were collected for cAMP assay.

RNase Protection Assay
For detection of the expression of different PDE4D variants in gonadotropin-treated rat ovaries, RNase protection assay (RPA) was performed using a RPAII Kit (Ambion, Inc., Austin, TX). The 276-bp probe common to all PDE4D transcripts and corresponding to the nucleotide 1634–1910 of rat PDE4D1 sequence was generated by PCR as previously described (34). The predicted protected fragments for PDE4D1, PDE4D2, and PDE4D3/D5 were 276 bp, 170 bp, and 291 bp, respectively. The single-stranded RNA transcripts were synthesized from each linearized template using a Transcription in Vitro System Kit (Promega Corp., Madison, WI) with 32P-uridine triphosphate. The 32P-labeled cRNA probes were purified by acrylamide gel electrophoresis and then hybridized with 20–30 µg of ovarian total RNA at 43 C overnight. After hybridization, samples were digested with RNase A before precipitation and electorphoresis on a 5% denaturing polyacrylamide gel. After electrophoresis, gels were exposed to RX film (Eastman Kodak Co., Rochester, NY) for 48–72 h at -80 C.

PDE Assay
Ovaries collected from gonadotropin-treated mice were homogenized in ice-cold buffer according to the procedure described previously (27). Aliquots of the homogenates were assayed for total PDE activity, as well as rolipram-insensitive PDE activity, by incubation in the absence or presence of 10 µM rolipram, a specific inhibitor of PDE4. The rolipram-sensitive PDE4 activity was obtained by subtracting the rolipram-insensitive activity from total activity. PDE activity was measured according to the method of Thompson and Appleman (50) as detailed previously. Each assay was normalized by protein concentration that was determined by a colorimetric assay (51).

Immunoprecipitation and Western Blot Analysis
Ovaries collected from gonadotropin-treated rats were homogenized in ice-cold buffer containing 25 mM HEPES, 150 mM NaCl, 5 mM EDTA, 0.5% Triton, and a protease inhibitor mixture (Roche, Palo Alto, CA). After centrifugation at 10,000 x g for 15 min at 4 C, supernatants were used for immunoprecipitation as previously reported. Protein from the pellet was eluted with PBS containing 1% sodium dodecyl sulfate (SDS) for 20 min at room temperature. The eluted samples were separated by electrophoresis using 8% (wt/vol) polyacrylamide gel. After transfer, polyvinylidene difluoride membranes (Millipore Corp., Bedford, MA) were blocked and hybridized with a monoclonal-PDE4D antibody (gift from ICOS Corp., Seattle, WA). Immunoreactive bands were detected by using ECL detection reagent (Amersham Pharmacia Biotech, Piscataway, NJ).

Test of Ovulation Rate
Immature (20–30 d, body weight 10–12 g) female littermates of the three genotypes were injected ip with 5 IU of PMSG to induce multiple follicle growth. After 46–48 h, the animals received ip injection with 5 IU of hCG to induce ovulation. Mice were killed by cervical dislocation at 24 h after hCG treatment, and the ovulated oocytes were retrieved from ampulla of the oviduct and counted.

Ovarian Histology
Ovaries collected from PMSG-treated mice were fixed in Bouin’s solution containing 20% formaldehyde for 6 h at room temperature. Fixed ovaries were dehydrated, paraffin embedded, and sectioned. Serial sections of the entire ovary were stained with hematoxylin and eosin, and then examined by light microscopy. The number of luteinized follicles were followed through the serial sections and expressed per mouse. Four mice were used for each genotype.

cAMP Assay
The cAMP accumulation of ovarian and granulosa cells was measured according to the procedure described previously (24). Briefly, the ovaries and granulosa cells were homogenized with 5% trichloroacetic acid (TCA) and 0.1% TCA, respectively, in 95% ethanol and water. After centrifugation, the supernatant was dried under vacuum and assayed for cAMP content. The protein concentration of the pellets was assessed by the Lowry method (52).

Northern Blot Analysis
Total RNA from ovaries was isolated using TRIzol Reagent solution (Invitrogen Corp., Carlsbad, CA). Twenty micrograms of total RNA were separated by electrophoresis on a 1.2% agarose gel containing formaldehyde and transferred to nylon membranes. After UV cross-linking and prehybridization, membranes were hybridized for 4 h at 68 C in an ExpressHybridization solution (CLONTECH Laboratories, Inc., Palo Alto, CA) with 32P-labeled cDNA probes. After hybridization, membranes were washed twice for 5 min at room temperature in 2x saline sodium citrate (SSC) and 0.05% SDS, followed by 20–40 min at 50 C in 0.1x SSC and 0.1% SDS. Membranes were then exposed using Kodak RX film at -80 C. For normalization of data, blots were stripped and rehybridized with a cDNA probe for mouse glyceraldehyde-3-phosphate-dehydrogenase (GAPDH).

In Situ Hybridization
Ovaries from PMSG-stimulated wild-type, heterozygous, and PDE4D KO mice were fixed in 4% paraformaldehyde for 6 h and incubated in 0.5 M sucrose overnight at 4 C. The ovaries were embedded in OCT (Tissue-Tek, Torrance, CA), cut into 10-µm sections, and mounted on Superfrost slides (Fisher Scientific, Pittsburgh, PA). Slides were postfixed in paraformaldehyde and treated with 0.2 M HCl, 2x SSC at 70 C, pronase E (Sigma), 2 mg/ml glycine, and then 0.1 M triethanolamine. Slides were then placed in 0.25% acetic anhydride dehydrated in ethanol (30–100%). A 387-bp murine P450scc cDNA subcloned in pBluescript vector (kindly provided by K. P. Parker, Department of Medicine and Pharmacology, Duke University, Durham, NC) was linearized by EcoRI and BamHI, and transcribed to synthesize [35S]-labeled RNA probes. Hybridization mixtures with antisense and sense RNA probes were added to the slide and incubated overnight at 50 C. Posthybridization washes consisted of RNase A treatment and decreasing concentrations of SSC washes. Hybridized slides were dehydrated and then dipped into NTB2 Emulsion (Eastman Kodak Co.), exposed for 2 d, developed, and then counterstained with Gill’s hematoxylin and eosin Y (0.5% wt/vol in ethanol). After counterstaining, tissues were cleared with xylene, mounted with Permount (Fisher Scientific), then visualized and photographed with an AxioCam (Carl Zeiss, Thornwood, NY).

Progesterone Assay
The blood of gonadotropin-treated mice was collected and centrifuged at 1500 x g at 4 C for 30 min, and the serum was assayed for progesterone using an RIA Kit (Diagnosis Systems Laboratories, Webster, TX).

RT-PCR and Southern Blot Analysis
For semiquantitative measurements of gene expression, total RNA was extracted from gonadotropin-treated ovaries using TRIzol solution, and RT-PCR and Southern blot analysis was performed according to the procedure described previously (12). Briefly, first-strand cDNA was synthesized from total RNA using oligo-deoxythymidine primer. Specific primers were used to amplify cDNAs; PR forward, 5'-CACAGTGGTGGATTTCATCC-3'; PR reverse, 5'-GCTGGGGGCTTGCACGGCAG-3'; PACAP forward, 5'-TGACCATGTGTAGCGGAGCAA-3'; PACAP reverse, 5'-CGTCCTTTGTTTTTAACCCT-3'; COX-2 forward, 5'-TGTACAAGCAGTGGCAAAGG-3', COX-2 reverse, 5'-GCTGTGGATCTTGCACATTG-3'; GAPDH forward, 5'-GAAGGTCGGTGTGAACGGATTTGGC-3' and 5'CATGTAGGCCATGAGGTCCACCAC-3'. PCR products were fractionated by agarose gel electrophoresis and then transferred to a nylon membrane. Blots were hybridized with [{gamma}-32P]ATP-labeled oligonucleotide probes corresponding to nucleotide sequences nested between the specific PCR primers. The membranes were then washed in 1.5 x SSC/0.1% SDS at 48 C followed by exposure to x-ray film.

Data Analysis
Statistical significance was assessed by either t test or two-way ANOVA, and P < 0.05 was considered significant.


   ACKNOWLEDGMENTS
 
The authors are indebted to Dr. Alex Tsafriri for helpful discussions and Caren Spencer for editorial assistance.


   FOOTNOTES
 
1 J.-Y.P. and F.R. contributed equally to the study. Back

This work was supported by NIH Grant R01-HD-20788.

Abbreviations: COX-2, Cyclooxygenase-2; GAPDH, glyceraldehyde-3-phosphate-dehydrogenase; Gs, stimulatory guanine nucleotide binding protein; hCG, human chorionic gonadotropin; KO, knockout; P450scc, P450 side chain cleavage; PDE, phosphodiesterase; PMSG, pregnant mare serum gonadotropin; RNase, ribonuclease; RPA, RNase protection assay; SDS, sodium dodecyl sulfate; SSC, saline sodium citrate; TCA, trichloroacetic acid.

Received for publication December 20, 2002. Accepted for publication March 12, 2003.


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