Alexander V. Sirotkin “The role of protein kinases in control of ovarian functions”

Protein kinases are presented in the ovary

Presence of members of MAPK, CDK, TK, PI3K, Akt, PKA, AK, HER kinases, mTOR and activin receptor like kinase (ALK) families in ovarian follicular granulosa cells has been well documented (Sirotkin et al., 2003; Hunzicker-Dunn and Maizels, 2006; Kumar et al., 2007; Sawada et al., 2007; Wang and Tsang, 2007; Dupont et al., 2008; Makarulla et al., 2008).  Studies of effects of regulators of PKs indicate involvement of a number of other  PKs in control of ovarian functions and in mediating effect of hormones on these functions (see below).  Expression of members of MAPK (Meidan et al., 2005), PKC (Gadsby et al., 2006) and PKA  (Dupont et al., 2008) families was demonstrated in ovarian Corpus luteum.  PKs of PKA, MAPK, PI3K, Akt, CaMK, PKC, AK, Janus kinases (JNKs) and myosin light chain kinase (MLCK) were found in oocytes. Their expression and localization depends on stage of oocyte maturation suggesting their involvement in control of oogenesis (Ducibella and Fissore, 2008; Mermillod et al., 2008). 
PKs is affected by some reproductive disorders indicating involvement of these PKs in control of these disorders (see below). For example, in ovarian tumors, substantial changes (mainly overexpression of PK or its mRNA compared to healthy ovaries) in members of MAPK, TK, PK3K, Akt,  HER kinases, TOR a.o. families are described (Martin and Schilder, 2006; Kumar et al., 2007; Markman, 2008). Polycystic ovarian syndrome (PCOS) is associated with increased expression of MAPK/ERK1,2 and insulin receptor Ser kinase but reduced expression of PI3K and insulin receptor TK (Seow et al., 2007).
Expression of PKs in the ovary can be affected by hormones. For example, FSH activates some ovarian genes encoding PKs, their regulators or targets (Hunzicker-Dunn and Maizels, 2006). Influence of FSH and other hormones on ovarian members of PKA, MAPK, CDK, PI3K, Akt, CaMK, PKC and other PKs or their mRNAs (Gadsby et al., 2006; Hinzucker-Dunn and Maizels, 2006; Dupont et al., 2008; Mermillod et al., 2008) has been reported. It suggest, that PKs can be intracellular mediators of hormones action on ovarian cells (see below).

Protein kinases control ovarian cell proliferation

The genomic screen of PKs, which blockade by siRNAs affected human granulosa cell proliferation showed, that  36% of PKs  is involved in stimulation, but only 8% of PKs in inhibition of ovarian follicular cell cycle (Sirotkin et al., 2009).  Effect of pharmacological activators or blockers of PKs showed, that PKA, PKG, MAPKs, PI3K/Akt, CDKs, TKs and ALK can both promote (in some cases also suppress) ovarian granulosa cell proliferation (Sirotkin et al., 2003; Wang and Tsang, 2007).  The cAMP/PKA-dependent intracellular mechanisms can stimulate (Viegas et al., 2008) or not influence (Dupont et al., 2008) follicular cell proliferation, but other PKs are involved predominantly in promotion of ovarian cell cycle. The involvement of these and some other PKs in control of ovarian cell proliferation was confirmed by experiments with ovarian malignant cells. Pharmacological, siRNA- or antisense oligonucleotide-induced inhibition of PKC, PI3K, MAPK/MDM2, TKs including EGFR, ALK7, TOR, AK, and embryonic leucine zipper kinase (Melk) can inhibit proliferation of ovarian carcinoma cells and  reduce the growth of ovarian tumors (Martin and Schilder, 2006; Suga et al., 2007).
During Corpus luteum development, MAPKs can be involved in stimulation of luteal cell proliferation (Meidan et al., 2005).

Protein kinases control ovarian cell apoptosis

Genome-wide selected blockade of PKs by siRNAs showed, that  34% of PKs  up-regulates, and less than 6% of PKs down-regulate human ovarian granulosa cell apoptosis  (Sirotkin et al., 2009).  MAPKs, PI3K/Akt, CDKs, TKs, ALK and AK probably inhibit this process (Sirotkin et al., 2003; Wang and Tsang, 2007; Macarulla et al., 2008), and PKG stimulates it (Sirotkin et al., 2003). cAMP/PKA can either suppress (Vigas et al., 2008) or not affect (Sirotkin et al., 2003; Sirotkin, 2005) ovarian cell apoptosis.
On the contrary, genomic blockade of  PKs in ovarian carcinoma cells revealed the pro-apoptotic action of PI3K/Akt system (Noske et al., 2007), MAPK/MDM2  (Suga et al., 2007), TK/EphB4 (Kumar et al., 2007), TK/c-Met (Sawada et al., 2007), ALK7 (Xu et al., 2006) and Melk (Gray et al., 2005), but not of AK (Macarulla et al., 2008). Inhibition of TOR promotes apoptosis in ovarian carcinoma cells (Martin and Schilder, 2006).
In Corpus luteum, activation of PKC in mid-luteal phase can increase sensitivity of luteal cells to prostaglandin F2 alpha, which in turn initiate their apoptosis and subsequent luteolysis (Diaz et al., 2002; Gadsby et al., 2006).

Protein kinases control maturation of oocyte-cumulus complex

A number of PKs - PKA, PKG, MAPKs, PI3K, CDKs, TKs including EGFR, PKC, CaMK and MLCK - can be involved in control oocyte maturation and cytokinesis. MAPKs, CDKs, PI3K, Akt, CaMK , MLCK  and EGFR are promoters of meiosis resumption and progression, but other TKs and PKC seems to inhibit these processes. cAMP/PKA can either promote or suppress  oocyte maturation (Brunet and Maro, 2005; Ducibella and Fissore, 2008; Dupont et al., 2008 ; Mermillod et al, 2008).
Oocyte nuclear maturation (meiosis) is arrested by cytostatic factor (CSF) and promoted by maturation, meiosis or mitosis promoting factor (MPF), a complex of  CDK/p34 and cyclin B1. MAPKs, PI3K, Akt, Janus kinases, AK and CaMKs can  affect production, action and stability of both MPF and CSF  (Brunet and Maro, 2005). AK seems to be a promoter of phosphorylation cascade leading to MPF activation (Mermillod et al., 2008).  AMP/PKA can prevent oocyte maturation via phosphorylation/inactivation of CDK1, a key component of  MPF (Dupont et al., 2008). In adition, Akt, JNK and AK, whose are activated during oocyte maturation, might be related to cytoplasmic oocyte maturation, i.e. accumulation and phosphorylation of proteins, mRNAs and other molecules and structures required for further fertilization and early embryo development (Mermillod et al., 2008).

Protein kinases control release of hormones by ovarian cells

The genome-wide search for PKs controlling steroid hormones release demonstrated, that 39% of PKs promotes and 14% of PKs inhibits progesterone  release by human granulosa cells . 10% of PKs stimulates, and 19% of PKs inhibites the IGF-I output (Sirotkin et al., 2010).
Experiments with PKA blockers demonstrated, that PKA can promote release of oxytocin, IGFBP-3, PGF2 alpha, IGF-I, progesterone and estradiol (Sirotkin et al., 2003), although inhibitory action of  PKA on ovarian progesterone, testosterone and estradiol release by mammalian ovarian follicular cells has been reported too (Dupont et al., 2008). 
Effects of cGMP analogues showed, that PKG can be involved in stimulation of ovarian granulosa cell progesterone, and inhibition of IGF-I, oxytocin release (Sirotkin et al., 2003).
Action of TKs blockers demonstrated, that TKs can inhibit progesterone, estradiol, but not  IGF-I and PGF-2 alpha output by mammalian granulosa and whole ovarian follicles.  Our first studies did not reveal influence of MAPKs blockers on mammalian ovarian PGF2 alpha, PGE2 and OT secretion, but subsequent experiments demonstrated, that MAPKs can up-regulate ovarian PGF alpha and PGE2 output (Sirotkin et al., 2003).
The involvement of TK/EGFRK in promotion of progestagen release by mammalian ovarian cells has been reported (Motola et al., 2006).
PKA promotes progesterone release by large luteal cells. PKCs, on the contrary, inhibits progesterone release and maintains luteal prostaglandin 2 alpha release (Diaz et al., 2002; Niswender, 2002) .

Protein kinases control hormone receptors and response to hormones

Simulatory effect of cAMP/PKA, as well as an inhibitory action of cGMP/PKG (LaPolt et al., 2003; Sirotkin et al., 2003) on the expression of ovarian LH receptors was reported. Stimulators of cAMP/PKA can increase the responsibility of ovarian cells to Gn-RH (Sirotkin et al.,2003) and gonadotropins (Masciarelli et al., 2004; McKenna et al., 2005; Hinzucker-Dunn and Maizels, 2006). Moreover,  pharmacological regulators of cAMP/PKA can modify response of ovarian cells to GH (Sirotkin, 2005), IGF-I, IGF-II, EGF, oxytocin (Sirotkin et al., 2003) and progestins (Peluso, 2006). 

Protein kinases mediate effect of hormones on the ovary

It is  shown, that FSH can promote ovarian folliculogenesis, oocyte maturation and estrogen release via PKA, PI3 kinase,  protein kinase B(Akt),  CDKs,  S10 histone H3 kinases, p38 MAPK, ERK1,2 MAPK, ribosomal S6 protein kinase (p70S6K), stress-activated protein kinases (MSK), p21-activated protein kinase, or aurora kinase B, glucocorticoid kinase (SGK), and the Ser/Thr kinase mammalian target of rapamycin (mTOR) a.o. (Hunzicker-Dunn and Maizels, 2006; Ducibella and Fissore, 2008; Dupont et al., 2008). LH promotes release of luteal progesterone and prostaglandin F 2 alpha through PKA- and PKC-dependent intracellular mechanisms (Diaz et al., 2002; Niswender et al., 2002). GnRH can promote oocyte maturation and progesterone release via EGFRK (Motola et al., 2006). GH (Sirotkin, 2005) and oxytocin (Sirotkin et al., 2003) can prevent apoptosis and promote release of ovarian hormones via PKA and MAPKs. IGF-I, IGF-II and EGF can activate ovarian cell proliferation and secretory activity through cAMP/PKA, cGMP/PKG, MAPK-, PI3/Akt and glycogen synthase kinase (GSK) - dependent pathways (Sirotkin et al., 2003; Dupont et al., 2008). The stimulatory effects of these growth factors on oocyte maturation  (Sirotkin et al., 2003; Dupont et al., 2008) are mediated through PKA, MAPKs, CDKs and EGFRK. TNF alpha induces Corpus luteim regression through PKC (Gadsby et al., 2006). Progesterone prevents apoptosis, and promotes proliferation and survival of ovarian granulosa cells via the PKG-dependent pathway (Peluso, 2006).

Protein kinases can be used for control of fertility, ovarian cycle and health

Despite a demonstration of  PKs involvement in control of different ovarian functions, only few attempts were previously made to use it in practice for control of animal or human fertility. The reported attempts concerned exclusively PKA.
Mice, deficient in cAMP-specific phosphodiesterases – activators of PKA, have impaired differentiation of ovarian cells, their response to gonadotropin, oocyte maturation, ovulation and fertility (Masciarelli et al., 2004).  On the other hand, administration of PKA activators increased the number of ovulations, embryos and born pups in rats (McKenna et al., 2005), number of ovulation, ovulated oocytes and developed embryos in rabbits (Sirotkin et al., 2008a), as well as the number of fertilized oocytes in womens suffered from PCOS (Dupont et al., 2008). Therefore, regulators of PKA could be potentially useful for improvement of reproductive processes.
Regulators of PKA, PKG, MAPKs, PI3K, Akt, CDKs, TKs and ALK affecting ovarian follicular cell proliferation can be used for stimulation of ovarian folliculogenesis, increase in number of ovulations and, perhaps, for control of hypogonadism, PCOS and other ovarian disorders associated with impaired follicular growth.  Molecules targeting PKs controlling Corpus luteum cell proliferation (MAPKs), apoptosis and response to hormonal inductors of luteolysis (PKCs) could be potentially useful for control of luteal phase of ovarian/menstrual cycle, fertility, synchronisation of cycles in animal and human assisted reproduction, as well as for treatment of persistent Corpus luteum or other other disorders of the ovarian cycle. Regulators of PKA, PKG, MAPKs and CDKs, which can increase formation of receptors to hormonal stimulators and responsibility of ovarian cells to these stimulators  can be useful for improvement of hormone-mediated induction of ovulation and superovulation in assisted reproduction, treatment of infertility or, on the contrary, for improvement of  hormonal contraception. Alteration in  PKA, PKG, MAPKs, PI3K, CDKs, TKs including EGFRK, PKC and CaMK within the oocytes which could affect their maturation  could be applicable in animal and human in-vitro maturation and fertilization protocols for induction of oocyte nuclear maturation, synchronization of nuclear and cytoplasmic maturation or, on the contrary, as potential contraceptive agents. PKA, PKG, MAPKs, PI3K/Akt, CDKs, TKs including EGFK, which are regulators as well as mediators of ovarian hormones, could be used for  regulation of  either hormone-dependent reproductive events and for prevention of hormone-dependent disorders including hormone-related malignant transformation. Ovarian cancer is one of frequently appeared and dangerous reproductive disorder.  It was mentioned previously, that suppression of PKC, PI3K, MAPK/MDM2  TKs, ALK7  including EGFR, TOR, AK  and Melk can reduce  viability and proliferation of ovarian carcinoma cells, their response to hormonal stimulators of their malignancy (steroid hormones and growth factors), as well as in increase of their responsibility to treatments. The PKs listed above and their regulators could be used for prediction, prevention and treatment of ovarian cancer.
Therefore, PKs and their regulators could be potentially used for characterization, prediction and control of ovarian folliculogenesis and atresia, Corpus luteum functions, oocyte maturation, fertility, release of hormones, response of ovarian structures to hormonal regulators, as well as for treatment of some reproductive disorders. Therefore, PKs and their pharmacological and genomic regulators could be potent and promising tool in assisted reproduction, biotechnology, human and veterinary medicine, although the application of PKs in control of reproduction is just starting now.