Vertebrate ovaries represent a vigorously dynamic structure due to the constant change in follicles over time, and such structural changes in the ovaries are closely associated with remodeling of the extracellular matrix (ECM). In particular, ECM remodeling underlies major changes during ovulation, which denotes the shedding of one or more viable oocytes from fully grown ovarian follicles into the reproductive tract. Ovulation is triggered by a surge in gonadotropin luteinizing hormone (LH) in all vertebrates [1
]. It is widely recognized that follicle rupture during vertebrate ovulation involves proteolytic degradation at the apex of ovulating follicles. Using the teleost medaka, a suitable model for ovulation studies, proteolytic processes accompanying follicle rupture during ovulation have been explored [5
]. Our previous studies demonstrated that activation of the plasminogen activator/plasmin (Plau/Plasmin) system and matrix metalloproteinase (Mmp) system is required for the hydrolysis of ECM proteins present in the follicle layers of ovulating follicles [6
]. Upon activation of the Plau/Plasmin system, active plasmin is generated by proteolytic processing of the liver-derived precursor plasminogen by the follicle-produced active urokinase-type plasminogen activator-1 (Plau1). Plasmin, thus generated, is responsible for hydrolyzing laminin, a major ECM component of the basement membrane [7
]. After the Plau/Plasmin system is kept active for several hours, another proteolytic system involving three distinct Mmp enzymes, membrane type 1-Mmp (Mt1-mmp/Mmp14), membrane type 2-Mmp (Mt2-mmp/Mmp15), and gelatinase A (Mmp2), becomes active. Mmp2 activated proteolytically by Mmp14 hydrolyzes type IV collagen, another principal component of the basement membrane, while Mmp15 degrades the type I collagen most abundantly present in the theca cell layer [6
]. During these hydrolytic processes, plasminogen activator inhibitor-1 [8
] and the tissue inhibitor of metalloproteinase-2b (Timp2b) [6
] are involved in regulating the Plau/Plasmin system and Mmp system, respectively. Among the proteases involved in follicle layer ECM hydrolysis, Mmp15 is the only LH-inducible enzyme. Our recent study demonstrated that LH-induced expression of the mmp15
gene is accomplished in two steps. In the first step, nuclear progestin receptor Pgr is induced by the LH surge, and the resulting Pgr is then complexed with 17α, 20β-dihydroxy-4-pregnen-3-one (17,20βP)—the physiological progestin ligand for medaka Pgr [10
]—to become an active transcription factor. In the second step, activated Pgr, together with the transcription factor CCAAT/enhancer-binding protein β (Cebpb), contributes to the expression of mmp15
In our attempt to search for genes/proteins involved in the expression of mmp15 mRNA, we found that the cyclin-dependent protein kinase (CDK) inhibitor roscovitine inhibited not only follicle ovulation, but also the follicular expression of mmp15 mRNA in the medaka, implicating CDK in the expression of the protease gene in the follicle that is destined to ovulate. In this study, we suggest that after phosphorylation, Pgr becomes a functional transcription factor for mmp15 gene expression, and that Cdk9 and cyclin I (Ccni) are involved in the process of Pgr phosphorylation.
Follicle rupture during ovulation in vertebrates involves well-regulated ECM degradation at the apical region of ovulating follicles. Our previous studies using the teleost medaka showed that two distinct proteolytic enzyme systems, the Plau/plasmin system and the MMP system, contribute to follicle rupture. Among the proteolytic enzymes involved in the process, the role of Mmp15 is of particular interest, in that it serves as a protease that hydrolyzes collagen type I, a major ECM protein in the follicle layer of ovulating follicles, and that its expression is drastically induced in the granulosa cells of follicles at ovulation [6
]. We recently reported the involvement of at least two transcription factors, Pgr and Cebpb, in the expression of the mmp15
]. The current study was initiated following the finding that the CDK inhibitor roscovitine suppressed in vitro follicle ovulation, and the results of this study provide additional information on the regulatory mechanism for mmp15
expression occurring in the granulosa cells of ovulating follicles. Our new findings are as follows: (i) Pgr is phosphorylated, (ii) the Ccni–Cdk9 complex may be involved in Pgr phosphorylation, and (iii) Ccni is induced by the ovulatory LH surge.
In the present study, we demonstrated that medaka Pgr undergoes phosphorylation prior to its binding to the promoter region of the mmp15
gene. Electrophoretic analyses of Pgr revealed that the protein is phosphorylated at various sites, as indicated by the appearance of multiple polypeptide bands with a molecular mass greater than that of unphosphorylated Pgr. Our data also suggest that Pgr phosphorylation is largely due to the action of Cdk, because almost all phosphorylated Pgr bands disappeared upon roscovitine treatment of the follicle. Treatment with phosphatase, rescovitine, or CDK9-inhibitor II resulted in no apparent increase in unphosphorylated Pgr. In a separate experiment, we found that the phosphorylated form of Pgr was estimated to be approximately 20% of the total Pgr. Since the proportion of phosphorylated Pgr to total Pgr is low, we presume that changes in the Pgr protein level could not be clearly observed after phosphatase or inhibitor treatment. Previous studies documented that, like medaka Pgr, the human counterpart is also phosphorylated [22
]. Three PGR isoforms (generally known as PR-A, PR-B, and PR-C) are known in humans, and two major protein kinases involved in human PGR phosphorylation are reported to be MAPK and CDK2. Furthermore, serine residues that are phosphorylated by these protein kinases have been identified. Sequence homologies in fish Pgr and human PGR are very low, hence, a prediction of possible phosphorylation sites in medaka Pgr by searching conserved serine residues between the fish and human proteins was fruitless. Therefore, phosphorylation sites of medaka Pgr were predicted using the free program KinasePhos (http://kinasephos.mbc.nctu.edu.tw/
). With this prediction tool, Ser208 and Ser250 were shown to be phosphorylated by Cdk. In addition, Thr14, Thr143, Ser208, and Thr212 could be possible phosphorylation sites for MAPK, although our current data indicated that MAPK has little, if any, effect on Pgr phosphorylation in the fish ovulating follicle. Further studies are needed to establish the validity of such predictions in the future.
We showed that Cdk9 may be responsible for the phosphorylation of Pgr. This idea is supported by the fact that (i) Cdk9 is expressed in the granulosa cells of ovulating follicles, as demonstrated by both immunohistochemical and western blot analyses; (ii) similar to roscovitine, treatment of follicles with the CDK9 inhibitor consistently inhibited follicle ovulation, Pgr phosphorylation, and Mmp15 expression; (iii) Cdk9 knockout experiments using OLHNI-2 cells stably expressing Pgr resulted in a reduction of Pgr phosphorylation; and more importantly, (iv) Cdk9 forms a complex with Ccni in the follicle layers of ovulating follicles. In the context of Pgr phosphorylation, the involvement of Cdk1 is less likely, because the Cdk1 protein is lacking in granulosa cells of ovulating follicles despite the presence of a large abundance of the corresponding mRNA in these cells. Considering that Cdk1 is abundantly detected immunohistochemically in small-growing follicles, Cdk1 may play a role in the early stage of follicle growth. We previously found that, similar to cdk1
, transcripts of enteropeptidase [15
] and melatonin receptor subtype 1c [17
] were expressed in the fish ovary, but their translation products were not detectable. Direct evidence for the involvement of CDK9 in Pgr phosphorylation in the granulosa cells of ovulating follicles is needed in future studies.
Interestingly, CDK9-inhibitor II treatment of preovulatory follicles inhibited ovulation, while roscovitine showed inhibitory effects on both ovulation and GVBD of the follicles. This finding indicates that CDKs other than CDK9 may be involved in GVBD, an LH-induced event occurring in the oocyte of medaka.
Ccni was the only cyclin expressed in the granulosa cells of ovulating follicles. Therefore, we tentatively assume that Ccni serves as the regulatory subunit of Cdk9 in the follicle, although Cdk9 activation by Ccni must be experimentally verified. Indeed, we found that Ccni was present in granulosa cells of the follicle in association with Cdk9 protein. Another important finding of the present study is that Ccni is an LH-inducible protein in cells of ovulating follicles. Interestingly, treatment of follicles with RU486, a Pgr antagonist, significantly reduced LH-induced ccni expression. This observation strongly suggests that Ccni is expressed in follicles in an LH- and Pgr-dependent manner.
Based on the present observations together with our previous findings [6
], we propose a schematic model for the role of Ccni/Cdk9 in the pathway leading to mmp15
/Mmp15 expression after the LH surge in follicles (Figure 8
). In this model, we focus on the events occurring in granulosa cells, because these cells have been documented to play a major role in fish ovulation [24
]. Roscovitine’s effects are also shown in this model. A variety of ovulatory reactions, including the activation of many ovulation-related genes, are evoked in cells expressing the LH receptor in response to the surge of LH, which occurs approximately 18 h before ovulation in the 24-h spawning cycle [14
]. Activation of the LH receptor by the LH surge immediately causes an increase in the intracellular cAMP concentration, which leads to the activation of Pgr. Pgr, thus synthesized, would then undergo phosphorylation at multiple sites, mainly via Cdk9. Cdk9, which is constitutively expressed in granulosa cells, should be activated by Ccni. Ccni is also induced in granulosa cells in response to LH stimulation. Our current data suggest that the expression of Ccni is driven by the 17,20βP-activated transcription factor Pgr, and that Pgr-dependent Ccni expression is initiated approximately 9 h before ovulation. Activated Cdk9 complexed with Ccni is capable of phosphorylating Pgr at various sites. The increase in phosphorylated Pgr levels begins at 9 h before ovulation in the granulosa cells of ovulating follicles, and such a situation lasts thereafter until the time of ovulation. Another transcription factor, Cebpb, which is also induced in an LH-dependent manner, is synthesized in granulosa cells of the follicle [13
]. At several hours preceding follicle rupture, both phosphorylated Pgr and Cebpb bind to the promoter region of the mmp15
gene for activation. The resulting translation product, Mmp15, is eventually expressed on the surface of granulosa cells. To substantiate the above hypothesis, further analyses using siRNA and knockout experiments targeting the ccni
genes are necessary.
The present study points to the role of Ccni/Cdk9 in the generation of phosphorylated Pgr, which serves as a functional transcription factor for the expression of mmp15
/Mmp15. However, as documented in studies using mammalian models, alterations in the transcriptional activities of target genes are some of the roles known for phosphorylated Pgr [22
]. This posttranslational modification of Pgr may also influence promoter specificity [25
], receptor turnover [26
], and nuclear association [27
]. Further studies are needed to comprehensively clarify the roles of phosphorylated Pgr in the follicle rupture process involving Mmp15 in medaka ovulation.
Another intriguing observation of the present study is that LH-induced Ccni synthesis itself was inhibited by roscovitine. This suggests the involvement of Cdk in the expression of Ccni in granulosa cells of ovulating follicles. Cdk9 is the only Cdk detectable in granulosa cells throughout the 24-h spawning cycle, therefore, we speculate that Cdk9 may contribute to the expression of Ccni. However, there are two important questions relevant to the LH-induced expression of Ccni: i) what molecular species of Ccn could be an activator of Cdk9? and ii) in which process is activated Cdk involved, i.e., transcriptional or translational events of the ccni gene? To solve these questions, further studies are required.
In summary, we showed that Pgr, a transcription factor required for the expression of mmp15, undergoes considerable phosphorylation in granulosa cells of ovulating follicles, and that only the phosphorylated form of Pgr likely functions as a potent transcription factor for mmp15 expression. Our data indicated that Ccni/Cdk9 may be responsible for Pgr phosphorylation. To our knowledge, this is the first study to demonstrate the involvement of Cdk in the process of follicle rupture during ovulation in vertebrates. The findings provided in this study help elucidate the whole process of ECM degradation that occurs in follicles during ovulation after the ovulatory surge of LH.