Protein kinases: Signaling molecules controlling ovarian functions

The present focus survey represents a review of current knowledge concerning involvement of protein kinases in control of basic ovarian functions in mammals. Ovarian cells produce a number of protein kinases, whose expression depends on type of cells, their state and action of hormones and other protein kinases. A number of protein kinases are involved in control of ovarian cell proliferation, apoptosis, oocyte maturation, hormone release, reception and response to hormones, as well as in mediating action of hormones on these ovarian functions. Protein kinases and their regulators could be 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.     

RNA interference and ovarian functions

RNA interference, a recently discovered new mechanism controlling gene expression via small RNAs, was shown to be involved in characterization and control of basic ovarian cell functions. The main classes of small RNAs, as well as their expression in ovaries have been described. Furthermore, the successful application of RNA interference for study and control of basic ovarian functions (proliferation, apoptosis, secretory activity, luteogenesis, oocyte maturation, and related ovarian cell malignant transformation) and production of recombinant proteins have been demonstrated. Application of RNA interference in reproductive biology and medicine can be successful in two main areas: (1) characterization and prediction of physiological and pathological state (association between particular small RNA and physiological or pathological processes), (2) application of small RNAs for regulation of reproductive processes and treatment of reproductive disorders or their particular indexes. Problems of improvement of small RNA delivery to target ovarian cells and potent RNA interference‐related approaches for treatment of ovarian disorders (especially of ovarian cancer) have been discussed. J. Cell. Physiol. 225: 354–363, 2010. © 2010 Wiley‐Liss, Inc.     

Cytokines: signalling molecules controlling ovarian functions

The present short review demonstrates an important role of different cytokines (colony stimulating factors, tumor necrosis factors, interleukins, anti-Mullerian hormone, inhibin, activin, follistatin, bone morphogenetic proteins, growth and differentiation factors) in the control of different ovarian functions - ovarian cell proliferation, apoptosis, folliculogenesis, luteogenesis, oogenesis, release of hormones, response to upstream hormonal regulators, fertility and, in some cases, in development of ovarian disorders. The possibility of practical application of these molecules for characterization, prediction and regulation of the ovarian state including treatment of ovarian disorders is demonstrated.     

Growth factors controlling ovarian functions

The present review demonstrates an important role of different growth factors (of insulin-like growth factors, epidermal growth factors, vascular endothelial growth factor, thrombopoietin, erytropoietin, hepatocyte growth factor, and growth factors of Hedgehog, Wnt and Notch families) in control of different ovarian functions--ovarian cell proliferation, apoptosis, folliculogenesis, luteogenesis, oogenesis, release of hormones, response to upstream hormonal regulators, fertility and, in some cases, in development of ovarian disorders. The possibility of practical application of these growth factors for characterization, prediction, and regulation the ovarian state is demonstrated.     

Role of protein kinases in prolactin signaling in bovine oocyte-cumulus complexes

Mechanisms of prolactin signal transduction in generative and somatic cells of mammalian ovarian follicles have been studied only to a small extent. In the present work, the involvement oftyrosine kinases and protein kinase C in mediating of the previously revealed modulating effects of prolactin on the nuclear maturation of bovine oocytes and the morphologic-functional state of surrounding cumulus cells was investigated in vitro. Tyrosine kinase inhibitor, genistein, was found to suppress the stimulating action of prolactin on the completion of oocyte nuclear maturation and cumulus expansion, whereas protein kinase C inhibitor, calpostin C, did not affect these hormonal effects. Furthermore, both genistein and calpostin C inhibited the inducing influence of prolactin on the proliferative activity of cumulus cells. At the same time the retarding action ofprolactin on destructive processes in cumulus cells was blocked only in the presence of calpostin C. The results of the study suggest that the stimulatory influence of prolactin on oocyte nuclear maturation and attendant cumulus expansion is achieved with the participation of tyrosine kinases, whereas the modulating action of the hormone on the functional state of cumulus cells depends on activation of not only tyrosine kinases, but also protein kinase C.     

Involvement of obestatin,cyclin-dependent kinase and protein kinase C in control of feline ovarian cell viability and hormones release

The aim of our in vitro study was to understand the role of obestatin, cyclin-dependent kinase (CDK) and protein kinase C (PKC) in the control of basic feline ovarian cell functions (viability, ovarian hormones release), as well as the role of protein kinases in mediating the effect of obestatin on these processes. For this purpose, we analyzed the effect of obestatin (0, 10 and 100 ng/mL) alone or in combination with CDK blocker olomoucine (100 ng/mL) or PKC blocker calphostin-c (100 ng/mL) on cultured feline ovarian fragments or granulosa cells. The release of progesterone (P4), testosterone (T) and estradiol (E2) by isolated ovarian follicular fragments were evaluated by ELISA. Granulosa cell viability was analysed using the Trypan blue exclusion test. It was observed that the addition of obestatin alone significantly increased the granulosa cell viability (at dose 100 ng/mL), promoted the release of P4 (at all doses added) and IGF-I (at dose 100 ng/mL) but decreased T (at all doses added). E2 output was below the detection limit in all groups. The addition of either olomoucine or calphostin-c reduced cell viability, P4, T and IGF-I release. Both olomoucine and caplhostin-c inverted the stimulatory effect of obestatin on granulosa cell viability and were able to prevent stimulatory action of obestatin on ovarian cell viability and on hormone and growth factor release and change it to an inhibitory action. These observations show that obestatin can directly regulate (mostly promote) basal feline ovarian cell functions (hormone release and viability). The inhibitory action of CDK and PKC blockers on these functions suggests, that both CDK and PKC can be promoters of ovarian cell viability and steroidogenesis in cats. Furthermore, the ability of both CDK and PKC to prevent olomoucine action demonstrates that obestatin action on the feline ovary could be mediated by these kinases.     

Protein kinase implication in prolactin signaling in bovine oocyte-cumulus complexes

The mechanisms of prolactin signal transduction in generative and somatic cells of mammalian ovarian follicles are poorly understood. In this work, participation of tyrosine kinases and protein kinase C in mediation of the previously revealed modulating effects of prolactin on the nuclear maturation of bovine oocytes and the morphologic and functional state of surrounding cumulus cells in vitro has been investigated. It was found that a tyrosine kinase inhibitor genistein suppresses the stimulating action of prolactin on the completion of oocyte nuclear maturation and cumulus expansion, whereas a protein kinase C inhibitor calpostin C does not affect the hormonal effect. Furthermore, both genistein and calpostin C inhibited the inducing influence of prolactin on the proliferative activity of cumulus cells. At the same time, the retarding action of prolactin on destructive processes in cumulus cells was blocked only in the presence of calpostin C. These results show that the stimulating influence of prolactin on oocyte nuclear maturation accompanied by cumulus expansion is achieved with participation of tyrosine kinases, whereas the modulating action of the hormone on the functional state of cumulus cells depends on activation both of tyrosine kinases and protein kinase C.     

Leptin directly controls proliferation, apoptosis and secretory activity of cultured chicken ovarian cells

The aim of our in-vitro experiments was to examine, whether leptin can directly control functions of avian ovarian cells and to outline potential intracellular mediators of its effects. Granulosa cells or fragments of ovarian follicular wall were cultured with leptin (0, 1, 10 or 100 ng/mL medium). The expression of peptides involved in apoptosis (TdT, bax, its binding protein, bcl-2, ASK-1 and p53), cell cycle-related peptides (PCNA and cyclin B1), release of hormones (progesterone, testosterone, estradiol, arginine-vasotocin), as well as the expression of protein kinases (PKA, MAPK/ERK1,2 and CDK/p34) in the ovarian cells were examined by using immunocytochemistry, TUNEL, SDS-PAGE-Western immunoblotting, EIA and RIA. It was found that leptin inhibited expression of all markers of cytoplasmic apoptosis (bax, ASK-1 and p53), stimulated expression of anti-apoptotic peptide bcl-2, but did not affect nuclear DNA fragmentation (TdT). Furthermore, leptin inhibited expression of PCNA (marker of S-phase of mitosis), but not of cyclin B1 (marker of G phase of cell cycle). Moreover, it promoted release of progesterone and estradiol, suppressed release of testosterone, but did not affect arginine-vasotocin. Finally, leptin inhibited expression of MAPK/ERK1,2 and CDK/p34 and stimulated expression of PKA. The present observations demonstrate that leptin can directly control basic chicken ovarian functions - inhibit cytoplasmic apoptosis and proliferation (S-phase, but not G-phases of mitosis), regulate secretory activity (release of steroids, but not nonapeptide hormone) and expression of MAPK, PKA and CDC2, which might be potential intracellular mediators of leptin action.     

Nongenomic steroid-triggered oocyte maturation: Of mice and frogs

Luteinizing hormone (LH) mediates many important processes in ovarian follicles, including cumulus cell expansion, changes in gap junction expression and activity, sterol and steroid production, and the release of paracrine signaling molecules. All of these functions work together to trigger oocyte maturation (meiotic progression) and subsequent ovulation. Many laboratories are interested in better understanding both the extra-oocyte follicular processes that trigger oocyte maturation, as well as the intra-oocyte molecules and signals that regulate meiosis. Multiple model systems have been used to study LH-effects in the ovary, including fish, frogs, mice, rats, pigs, and primates. Here we provide a brief summary of oocyte maturation, focusing primarily on steroid-triggered meiotic progression in frogs and mice. Furthermore, we present new studies that implicate classical steroid receptors rather than alternative non-classical membrane steroid receptors as the primary regulators of steroid-mediated oocyte maturation in both of these model systems.     

The effect of hormones and growth factor on the course of meiosis in murine oocytes in culture]

The role of some intraovarian regulators of the final stages of gametogenesis is analysed. It is shown that the epidermal growth factor (EGF) in concentration of 1 and 10 ng/ml is able to induce reinitiation of meiosis from dictyotene stage during cultivation of the ovarian follicles of prepuberal mice in the serum-free medium after gonadotrophic stimulation. The pattern of maturation was analogous to that of maturation after HCG (LH) administration. Also, the EGF is able to stimulate meiosis reinitiation in the culture of cumulus-free oocytes blocked with cAMP at the stage of dictyotene. At the same time fibroblast growth factors and insulin do not demonstrate such an activity. Taking into consideration a high sensibility of oocytes to the EGF action, and also the fact that the character of changes of steroid hormones secreted by the ovary in culture under the action of EGF is the same as that under the influence of LH it is suggested that, the EGF and EGF-like proteins secreted by somatic follicle cells are the paracrinic regulators of the mammalian oocyte maturation which modulate neuroendocrine factors of the oogenesis control.     

Ghrelin and obestatin can promote human ovarian granulosa cell functions and FSH effects

The aim of the present in-vitro experiments was to examine the direct influence of ghrelin and obestatin on viability, proliferation and progesterone release by human ovarian granulosa cells and their response to FSH administration. Human granulosa cells were cultured in presence of ghrelin or obestatin (both at 0, 1, 10 or 100 ng/ml) alone or in the presence of FSH (10 ng/ml). Cell viability, accumulation of proliferation markers PCNA and cyclin B1 and release of progesterone were analyzed by Trypan blue extrusion test, quantitative immunocytochemistry and ELISA. Ghrelin, obestatin and FSH up-regulated all the measured ovarian cell parameters. Moreover, both ghrelin and obestatin promoted all the stimulatory effects of FSH. The obtained results demonstrate the direct stimulatory action of ghrelin, obestatin and FSH on basic ovarian cell functions, as well as the ability of metabolic hormones to improve FSH action on human ovarian cells.     

Kisspeptin as autocrine/paracrine regulator of human ovarian cell functions: Possible interrelationships with FSH and its receptor

The present study aims to examine the role of kisspeptin (KP), FSH, and its receptor (FSHR), and their interrelationships in the control of basic human ovarian granulosa cells functions. We investigated: (1) the ability of granulosa cells to produce KP and FSHR, (2) the role of KP in the control of ovarian functions, and (3) the ability of KP to affect FSHR and to modify the FSH action on ovarian functions. The effects of KP alone (0, 10 and 100 ng/mL); or of KP (10 and 100 ng/mL) in combination with FSH (10 ng/mL) on cultured human granulosa cells were assessed. Viability, markers of proliferation (PCNA and cyclin B1) and apoptosis (bax and caspase 3), as well as accumulation of KP, FSHR, and steroid hormones, IGF-I, oxytocin (OT), and prostaglandin E2 (PGE2) release were analyzed by the Trypan blue exclusion test, quantitative immunocytochemistry, and ELISA. KP given at a low dose (10 ng/mL) stimulated viability, proliferation, inhibited apoptosis, promoted the release of progesterone (P4), estradiol (E2), IGF-I, OT, and PGE2, the accumulation of FSHR, but not testosterone (T) release. KP given at a high dose (100 ng/mL) had the opposite, inhibitory effect. FSH stimulated cell viability, proliferation and inhibited apoptosis, promoted P4, T, E2, IGF-I, and OT, but not PGE2 release. Furthermore, KP at a low dose promoted the stimulatory effect of FSH on viability, proliferation, P4, E2, and OT release, promoted its inhibitory action on apoptosis, but did not modify its action on T, IGF-I, and PGE2 output. KP at a high dose prevented and inverted FSH action. These results suggest an intra-ovarian production and a functional interrelationship between KP and FSH/FSHR in direct regulation of basic ovarian cell functions (viability, proliferation, apoptosis, and hormones release). The capability of KP to stimulate FSHR, the ability of FSH to promote ovarian functions, as well as the similarity of KP (10 ng/mL) and FSH action on granulosa cells’ viability, proliferation, apoptosis, steroid hormones, IGF-I, OT, and PGE2 release, suggest that FSH influence these cells could be mediated by KP. Moreover, the capability of KP (100 ng/mL) to decrease FSHR accumulation, basal and FSH-induced ovarian parameters, suggest that KP can suppress some ovarian granulosa cell functions via down-regulation of FSHR. These observations propose the existence of the FSH-KP axis up-regulating human ovarian cell functions.     

Effect of follicular cells,IGF-I and tyrosine kinase blockers on oocyte maturation

The aim of this study was to test the following hypotheses: (i) that oocyte maturation is controlled by surrounding follicular cells; (ii) that a meiosis-regulating factor of follicular origin is not species-specific; (iii) that one of the follicular regulators of oocyte maturation is IGF-I; and, (iv) that Cumulus oophorus and tyrosine kinase-dependent intracellular mechanisms do not mediate IGF-I action on oocytes. It was found that co-culture of cumulus-enclosed bovine oocytes with isolated bovine ovarian follicles or with isolated porcine ovarian follicles significantly increased the proportion of matured oocytes (at metaphase II of meiosis) after culture. Porcine oocytes without cumulus investments had lower maturation rates than cumulus-enclosed oocytes. Co-culture with isolated porcine ovarian follicles resulted in stimulation of maturation of both cumulus-free and cumulus-enclosed porcine oocytes. These observations suggest that follicular cells (whole follicles or Cumulus oophorus) support bovine and porcine oocyte maturation, and that follicular maturation-promoting factor is not species-specific. The release of significant amounts of IGF-I by cultured bovine and porcine isolated follicles and granulosa cells was demonstrated. Addition of IGF-I to culture medium at 10 or 100 (but not 1000) ng/ml stimulated meiotic maturation of both cumulus-enclosed and cumulus-free porcine oocytes. Neither of the tyrosine kinase blockers, genistein or lavendustin (100 ng/ml medium), changed the stimulating effect of IGF-I on porcine oocytes. The present data suggest that at least one of the follicular stimulators of oocyte nuclear maturation is IGF-I, and that its effect is probably not mediated by cumulus investment or by tyrosine kinase-dependent intracellular mechanisms.     

Gonadotropic control of ovarian follicle maturation: the two-stage concept and its mechanisms

Most research on the control of oocyte maturation by luteinizing hormone (LH) in teleosts and amphibians has focused on the production and action of maturation-inducing hormone (MIH), the follicular hormone that directly triggers the resumption of oocyte meiosis. However, current information indicates that LH regulates maturation in two stages, and that 'oocyte maturation' can be appropriately described within the broader context of 'ovarian follicle maturation'. During the first stage of maturation the follicle (somatic) cells acquire the ability to produce MIH and the oocyte to respond to MIH (i.e. oocyte maturational competence, OMC), whereas in the second stage the follicle cells produce MIH and, consequently, the oocyte is released from meiotic arrest. A number of factors such as insulin-like growth factor-I, serotonin, and others may mediate or modulate the OMC-inducing action of LH. Like the acquisition of MIH-producing ability, the acquisition of OMC requires activation of the protein kinase A pathway. Two major cellular events associated with OMC acquisition are increases in homologous and heterologous gap junction contacts and in oocyte MIH receptor activity. The increased oocyte MIH receptor activity is presumably associated with OMC acquisition, but the significance of changes in gap junction contacts is at present uncertain. To eliminate inconsistency and ambiguity associated with current terminology we propose that the term, ovarian follicle (or oocyte) maturation be used for teleosts without qualifiers such as 'final' to define the first and second stages of follicular maturation.     

Characterization and action of meiotic maturation inhibitors in starfish ovary

Mechanical release of oocytes from the ovary of the starfish Asterias amurensis into sea water results in “spontaneous” meiotic maturation of the oocytes. The substances blocking the maturation of Asterias oocytes have been purified from the ovary and shown to be steroid glycosides named asterosaponins A and B. The extract prepared from isolated oocytes was incapable of inhibiting oocyte maturation. The ovarian extract inhibited the production of 1-methyladenine (1-MA) in follicle cells surrounding the oocyte. The ovarian extract failed to influence 1-MA-induced maturation of the oocyte with or without follicle cells. It can be concluded from the present results that the role of the ovarian extract containing steroid glycosides is to arrest “spontaneous” production of 1-MA in follicle cells. The suppression can be overcome by the action of a gonadotropic peptide hormone released from the nerve tissue.     

The Hormonal Control of the Amphibian Ovary

The ultimate control of amphibian gonadal function rests withenvironmen tal factors mediated through the hypothalamus. Itappears that control of ovarian growth resides in the infundibularregion and ovulation in the preoptic area. For normal temporalrelationships between oocyte growth and ovulation to occur,an intact hypothalamo-pituitary complex is necessary. It isuncertain whether the several types of pituitary basophils considered,histologically as gonadotropin producing cells are in fact producingseparate LH and FSH like hormones. Perhaps the concensus indicatesa single hormone has both vitellogenic and ovulatory functions.This hormone stimulates estrogen synthesis and secretion bythe ovarian follicle cells, and this steroid causes oviductgrowth and the hepatic biosynthesis of vitellogenin, the majoryolk platelet precursor. Uptake of this lipoprotein from thecirculation and its conversion to the components of the plateletis mediated by the gonadotropin, the presence of which resultsin the establishment of a rapid micropinocytotic process atthe level of the oocyte surface and of a mechanism for crystallizationof the yolk. A sudden surge of pituitary hormone, when presentedto fully grown oocytes leads to their maturation and ovulation,and to oviducal jelly release in some species. The active hormoneis progestin in nature, again produced by the follicle cells. In this review the known factors involved in the hypothalamohypophysio ovarian axis are discussed together with some considerationof outstanding problemsand the possible relevance ot ovipantvand ovovivipanty in amphibians to the ovarian control foundin viviparous species.     

Inhibition of rat oocyte maturation and ovulation by nitric oxide: mechanism of action

Established gap junctional communication (GJC) in the ovarian follicle is essential for maintaining the oocytes in meiotic arrest. Alternatively, LH-induced reinitiation of meiosis is subsequent to breakdown of GJC. It was recently reported that nitric oxide (NO) inhibits maturation in rat follicle-enclosed oocytes and elevates GJC in cultured mesangial cells. Taking these observations into account, we hypothesized that NO prevents reinitiation of meiosis by antagonizing the effect of LH on GJC in the ovarian follicle. Indeed, we found that NO interferes with LH-induced disruption of GJC as well as with the decrease of the expression of the gap junction protein GJA1 (previously known as CONNEXIN43). We also demonstrated that NO prevents activation of LH-induced mitogen-activated protein kinases (MAPKs) 1 and 2 and inhibits cumulus expansion. Along this line, incubation of ovarian follicles with an inhibitor of soluble guanylate cyclase, which is a downstream NO effector, induced on its own oocyte maturation as well as cumulus expansion. Unlike previous studies, we show here that elevation of NO resulted in inhibition of ovulation. We conclude that the mechanism by which NO inhibits LH-induced oocyte maturation possibly involves a negative effect on MAPK activation and, in turn, interference with interruption of GJC. This action of NO in the ovarian follicle is apparently mediated by cGMP. In addition, the negative effect of NO on ovulation may be subsequent to its inhibitory effect on cumulus expansion. Together, this study suggests that the preovulatory decrease in NO concentrations is a prerequisite for the ovarian response to LH.     

Dichotomous effects of forskolin on somatic and germ cell components of the ovarian follicle: evidence of cAMP involvement in steroid production and action

The role of cyclic AMP (cAMP) in ovarian follicular functions in Rana pipiens was investigated with the use of the adenylate cyclase stimulator, forskolin, which is thought to elevate intracellular level of cAMP. Effects of forskolin on oocyte germinal vesicle breakdown (GVBD) and on progesterone production by the follicles were assessed during the course of in vitro culture. Addition of forskolin to culture medium suppressed both progesterone-and frog pituitary homogenate (FPH)-induced meiotic maturation of the oocytes. Inhibitory effects of forskolin were essentially reversible and forskolin completely inhibited GVBD when added during the first four hours of incubation following exposure to progesterone. Forskolin alone stimulated a low level progesterone production by isolated follicles, but markedly stimulated progesterone production when it was supplemented with a low dose of FPH (0.005 pituitary equivalent/ml). Thus, forskolin acts synergistically with FPH on follicle cells to stimulate progesterone production. A higher dose of FPH (0.05 pitui. eq./ml) produced no additional synergistic effect of forskolin. Therefore, forskolin appears to have two contradictory functions in ovarian follicles: it augments FPH induced follicle secretion of meiosis initiator, progesterone, and simultaneously suppresses the maturation of the oocytes triggered by exogenous progesterone or FPH. The data presented indicate that there are two independent adenylate cyclase systems in the ovarian follicles which have separate functions: one in the follicle cells and the other in the oocyte. The two enzyme systems are thus compartmentalized and regulate different biological functions using the same messenger, cAMP. The data provide evidence that in amphibians, as in mammals, pituitary hormones regulate steroid hormone production by follicle cells via a cyclic AMP system. Thus, control of oocyte maturation induction appears to be determined by the relative levels of cAMP present in the follicle cells and oocytes.