Luteinizing hormone signaling in preovulatory follicles involves early activation of the epidermal growth factor receptor pathway

LH activates a cascade of signaling events that are propagated throughout the ovarian preovulatory follicle to promote ovulation of a mature egg. Critical to LH-induced ovulation is the induction of epidermal growth factor (EGF)-like growth factors and transactivation of EGF receptor (EGFR) signaling. Because the timing of this transactivation has not been well characterized, we investigated the dynamics of LH regulation of the EGF network in cultured follicles. Preovulatory follicles were cultured with or without recombinant LH and/or specific inhibitors. EGFR and MAPK phosphorylation were examined by immunoprecipitation and Western blot analyses. By semiquantitative RT-PCR, increases in amphiregulin and epiregulin mRNAs were detected 30 min after recombinant LH stimulation of follicles and were maximal after 2 h. LH-induced EGFR phosphorylation also increased after 30 min and reached a maximum at 2 h. EGFR activation precedes oocyte maturation and is cAMP dependent, because forskolin similarly activated EGFR. LH-induced EGFR phosphorylation was sensitive to AG1478, an EGFR kinase inhibitor, and to inhibitors of matrix metalloproteases GM6001 and TNFalpha protease inhibitor-1 (TAPI-1), suggesting the involvement of EGF-like growth factor shedding. LH- but not amphiregulin-induced oocyte maturation and EGFR phosphorylation were sensitive to protein synthesis inhibition. When granulosa cells were cultured with a combination of neutralizing antibodies against amphiregulin, epiregulin, and betacellulin, EGFR phosphorylation and MAPK activation were inhibited. In cultured follicles, LH-induced MAPK activation was partially inhibited by AG1478 and GM6001, indicating that this pathway is regulated in part by the EGF network but also involves additional pathways. Thus, complex mechanisms are involved in the rapid amplification and propagation of the LH signal within preovulatory follicles and include the early activation of the EGF network.     

Luteinizing hormone-dependent activation of the epidermal growth factor network is essential for ovulation

In the preovulatory ovarian follicle, mammalian oocytes are maintained in prophase meiotic arrest until the luteinizing hormone (LH) surge induces reentry into the first meiotic division. Dramatic changes in the somatic cells surrounding the oocytes and in the follicular wall are also induced by LH and are necessary for ovulation. Here, we provide genetic evidence that LH-dependent transactivation of the epidermal growth factor receptor (EGFR) is indispensable for oocyte reentry into the meiotic cell cycle, for the synthesis of the extracellular matrix surrounding the oocyte that causes cumulus expansion, and for follicle rupture in vivo. Mice deficient in either amphiregulin or epiregulin, two EGFR ligands, display delayed or reduced oocyte maturation and cumulus expansion. In compound-mutant mice in which loss of one EGFR ligand is associated with decreased signaling from a hypomorphic allele of the EGFR, LH no longer signals oocyte meiotic resumption. Moreover, induction of genes involved in cumulus expansion and follicle rupture is compromised in these mice, resulting in impaired ovulation. Thus, these studies demonstrate that LH induction of epidermal growth factor-like growth factors and EGFR transactivation are essential for the regulation of a critical physiological process such as ovulation and provide new strategies for manipulation of fertility.     

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.     

Endocrine, paracrine and autocrine control of follicular development

Development of a single follicle during the menstrual cycle is under control of hormones stimulating follicular maturation, ovulation and luteogenesis. Several factors intervene locally to prevent other follicles from developing at the same time as dominant follicle. These other follicles remain quiescent or evaluate to atresia. Atresia results from the action of several endocrine, paracrine and autocrine mechanisms which synergistically inhibit aromatase activity. The subsequent lack of estrogens reduces granulosa cell multiplication. The oocyte will not become fertilizable before the preovulatory peak of LH, after the resumption of meiosis and after reaching metaphase of the second meiotic division. Several factors are involved in the inhibition of spontaneous resumption of meiosis: cyclic nucleotides, sex steroids, somatostatin and oocyte maturation inhibitor(s) (OMI). Ovulation is related to breakdown of connective tissue synthesized by granulosa cells under the influence of FSH. Connective tissue lysis is dependent on proteolytic enzymes which are released and activated by FSH, LH and relaxin. A paracrine control could be involved in ovulation: LH induces the production of prostaglandin and relaxin by theca cells which, in turn, stimulate collagenase and proteoglycanase secretion by granulosa cells.     

Luteinizing hormone reduces the activity of the NPR2 guanylyl cyclase in mouse ovarian follicles, contributing to the cyclic GMP decrease that promotes resumption of meiosis in oocytes

In preovulatory ovarian follicles of mice, meiotic prophase arrest in the oocyte is maintained by cyclic GMP from the surrounding granulosa cells that diffuses into the oocyte through gap junctions. The cGMP is synthesized in the granulosa cells by the transmembrane guanylyl cyclase natriuretic peptide receptor 2 (NPR2) in response to the agonist C-type natriuretic peptide (CNP). In response to luteinizing hormone (LH), cGMP in the granulosa cells decreases, and as a consequence, oocyte cGMP decreases and meiosis resumes. Here we report that within 20 min, LH treatment results in decreased guanylyl cyclase activity of NPR2, as determined in the presence of a maximally activating concentration of CNP. This occurs by a process that does not reduce the amount of NPR2 protein. We also show that by a slower process, first detected at 2h, LH decreases the amount of CNP available to bind to the receptor. Both of these LH actions contribute to decreasing cGMP in the follicle, thus signaling meiotic resumption in the oocyte.     

Role of the epidermal growth factor network in ovarian follicles

The LH surge causes major remodeling of the ovarian follicle in preparation for the ovulatory process. These changes include reprogramming of granulosa cells to differentiate into luteal cells, changes in cumulus cell secretory properties, and oocyte maturation. This review summarizes published data in support of the concept that LH stimulation of ovarian follicles involves activation of a local epidermal growth factor (EGF) network. A model describing this property of LH signaling and its branching to other signaling modules is discussed. According to this model, LH activation of mural granulosa cells stimulates cAMP signaling, which, in turn, induces the expression of the EGF-like growth factors epiregulin, amphiregulin, and betacellulin. These growth factors function by activating EGF receptors in either an autocrine/juxtacrine fashion within the mural layer, or they diffuse to act on cumulus cells. Activation of EGF receptor signaling in cumulus cells, together with cAMP priming, triggers oocyte nuclear maturation and acquisition of developmental competence as well as cumulus expansion. This model has important implications for ovarian physiology and for the development of new strategies for the pharmacological control of ovulation and for gamete maturation in vitro.     

哺乳动物卵母细胞减数分裂恢复的诱导

促使哺乳动物卵母细胞减数分裂恢复的机制尚不十分清楚。有腔卵泡中发育充分的卵母细胞被减数分裂抑制因子阻滞在生发泡（GV）期,环一磷酸腺苷（cAMP)是研究得最为清楚的减数分裂抑制因子。然而,其它因子也参与了卵母细胞减数分裂的阻滞。虽然排卵前的促黄体素（LH）峰诱导卵母细胞减数分裂恢复已成定论,但是参与该事件的各种过程非常复杂,因而还没有完全确定。目前,有两种主要但并不互相排斥的假说。第一种假说认为,LH对颗粒细胞的刺激作用终止减数分裂抑制因子流向卵母细胞,从而使卵母细胞膈离这些抑制因子并进而促使减数分裂恢复,第二种假设认为LH刺激颗粒细胞产生一种减数分裂诱导信号,该信号进而克服或者破坏减数分裂抑制因子的作用。权衡这两种假说,目前的证据倾向于支持阳性信号假说,而且最近的研究暗示,该种阳性信号的产生发生在颗粒细胞中LH诱导的cAMP水平上升和MAPK激酶激活之后。     

Actions of epidermal growth factor receptor/mitogen-activated protein kinase and protein kinase C signaling in granulosa cells from Gallus gallus are dependent upon stage of differentiation

Studies in both mammalian and nonmammalian ovarian model systems have demonstrated that activation of the mitogen-activated protein kinase (MAPK) and protein kinase C (PKC) signaling pathways modulates steroid biosynthesis during follicle development, yet the collective evidence for facilitory versus inhibitory roles of these pathways is inconsistent. The present studies in the hen ovary describe the changing role of MAPK and PKC signaling in the regulation of steroidogenic acute regulatory protein (STAR) expression and progesterone production in undifferentiated granulosa cells collected from prehierarchal follicles prior to follicle selection versus differentiated granulosa from preovulatory follicles subsequent to selection. Treatment of undifferentiated granulosa cells with a selective epidermal growth factor receptor (EGFR) and ERBB4 receptor tyrosine kinase inhibitor (AG1478) both augments FSH receptor (Fshr) mRNA expression and initiates progesterone production. Conversely, selective inhibitors of both EGFR/ERBB4 and MAPK activity attenuate steroidogenesis in differentiated granulosa cells subsequent to follicle selection. In addition, inhibition of PKC signaling with GF109203X augments FSH-induced Fshr mRNA plus STAR protein expression and initiates progesterone synthesis in undifferentiated granulosa cells, but inhibits both gonadotropin-induced STAR expression and progesterone production in differentiated granulosa. Granulosa cells from the most recently selected (9- to 12-mm) follicle represent a stage of transition as inhibition of MAPK signaling promotes, while inhibition of PKC signaling blocks gonadotropin-induced progesterone production. Collectively, these data describe stage-of-development-related changes in cell signaling whereby the differentiation-inhibiting actions of MAPK and PKC signaling in prehierarchal follicle granulosa cells undergo a transition at the time of follicle selection to become obligatory for gonadotropin-stimulated progesterone production in differentiated granulosa from preovulatory follicles.     

Regulation of the follicular hierarchy and ovulation

Studies are discussed which investigate the regulation of follicular maturation and the ovulation sequence of the domestic hen. The number of FSH receptors of ovarian granulosa cells decreases as the follicle matures, and this decrease in receptor number is paralleled by a gradual loss of FSH-stimulable adenylyl cyclase (AC) activity. By contrast, LH-stimulable AC activity increases as the follicle progresses through the hierarchy. In addition, FSH stimulates progesterone secretion by granulosa cells of the smaller preovulatory follicles, whereas these cells are only minimally responsive to LH. These data suggest that the maturation of less mature (smaller) follicles is primarily controlled by FSH, while LH may serve primarily as the ovulation-inducing hormone. The ability of LH to stimulate progesterone release and induce premature ovulation is dependent upon the stage of the sequence. Injection of ovine LH 12 hr prior to ovulation of the first (C1) egg of the sequence induces fully potentiated preovulatory plasma progesterone surges and 100% premature ovulation, whereas injection prior to the second (C2) ovulation of the sequence fails to stimulate prolonged progesterone release and induces premature ovulation in less than 50% of injected hens. These results are consistent with data obtained in vitro which suggest that granulosa cells obtained 12 hr prior to a C1 ovulation secrete more progesterone in response to chicken LH compared to those obtained 12 hr prior to the C2 ovulation. These data are discussed in terms of the ovary's ability to act as a regulator of the ovulatory cycle.     

GnRH actions on rat preovulatory follicles are mediated by paracrine EGF-like factors

Gonadotropin releasing hormone (GnRH) has been shown to mimic the actions of LH/hCG on oocyte maturation and ovulation. Recent studies demonstrated that induction of ovulation by LH/hCG is mediated, at least in part, by transactivation of epidermal growth factor receptors (EGFR) by autocrine/paracrine EGF-like factors activated by metalloproteases. Here we have examined whether the action of GnRH on the preovulatory follicles is exerted through similar mechanisms involving activation of EGFR. The EGFR kinase inhibitor, AG1478, inhibited GnRH-induced oocyte maturation in explanted follicles in vitro. Its inactive analog, AG43, did not affect GnRH-stimulated resumption of meiosis. GnRH, like LH, stimulated transient follicular expression of EGF-like agents, as well as rat cycloxygenase-2 (rCOX-2), rat hyaluronan synthase-2 (rHAS-2), and rat tumor necrosis factor-alpha-stimulated gene 6 (rTSG-6) mRNAs, known ovulatory enzymes. Likewise, GnRH stimulated follicular progesterone synthesis. Conversely AG1478 inhibited all these actions of GnRH. Furthermore, Galardin, a broad-spectrum metalloprotease inhibitor, blocked GnRH-induced oocyte maturation and follicular progesterone synthesis. In conclusion, we have demonstrated that follicular EGF-like factors mediate also the GnRH-stimulation of ovulatory changes, like these of LH/hCG.     

Follicle-restricted compartmentalization of transforming growth factor beta superfamily ligands in the feline ovary

Ovarian follicular development, follicle selection, and the process of ovulation remain poorly understood in most species. Throughout reproductive life, follicle fate is balanced between growth and apoptosis. These opposing forces are controlled by numerous endocrine, paracrine, and autocrine factors, including the ligands represented by the transforming growth factor beta (TGFbeta) superfamily. TGFbeta, activin, inhibin, bone morphometric protein (BMP), and growth differentiation factor 9 (GDF-9) are present in the ovary of many animals; however, no comprehensive analysis of the localization of each ligand or its receptors and intracellular signaling molecules during folliculogenesis has been done. The domestic cat is an ideal model for studying ovarian follicle dynamics due to an abundance of all follicle populations, including primordial stage, and the amount of readily available tissue following routine animal spaying. Additionally, knowledge of the factors involved in feline follicular development could make an important impact on in vitro maturation/in vitro fertilization (IVM/IVF) success for endangered feline species. Thus, the presence and position of TGFbeta superfamily members within the feline ovary have been evaluated in all stages of follicular development by immunolocalization. The cat inhibin alpha subunit protein is present in all follicle stages but increases in intensity within the mural granulosa cells in large antral follicles. The inhibin betaA and betaB subunit proteins, in addition to the activin type I (ActRIB) and activin type II receptor (ActRIIB), are produced in primordial and primary follicle granulosa cells. Additionally, inhibin betaA subunit is detected in the theca cells from secondary through large antral follicle size classes. GDF-9 is restricted to the oocyte of preantral and antral follicles, whereas the type II BMP receptor (BMP-RII) protein is predominantly localized to primordial- and primary-stage follicles. TGFbeta1, 2, and 3 ligand immunoreactivity is observed in both small and large follicles, whereas the TGFbeta type II receptor (TGFbeta RII) is detected in the oocyte and granulosa cells of antral follicles. The intracellular signaling proteins Smad2 and Smad4 are present in the granulosa cell cytoplasm of all follicle size classes. Smad3 is detected in the granulosa cell nucleus, the oocyte, and the theca cell nucleus of all follicle size classes. These data suggest that the complete activin signal transduction pathway is present in small follicles and that large follicles primarily produce the inhibins. Our data also suggest that TGFbeta ligands and receptors are colocalized to large antral follicles. Taken together, the ligands, receptors, and signaling proteins for the TGFbeta superfamily are present at distinct points throughout feline folliculogenesis, suggesting discrete roles for each of these ligands during follicle maturation.     

Induction of maturation in follicle-enclosed oocytes: the response to gonadotropins at different stages of follicular development

Antral follicles, isolated from either nontreated or pregnant mare's serum gonadotropin (PMSG)-primed 27-day-old rats, were incubated in the absence or the presence of either luteinizing hormone (LH), follicle-stimulating hormone (FSH), or forskolin. The effect of these agents on oocyte maturation and cyclic adenosine 3',5'-monophosphate (cAMP) accumulation was studied and compared. Both gonadotropins, LH and FSH, as well as forskolin, effectively induced maturation of oocytes enclosed by large antral follicles isolated from PMSG-primed rats. On the other hand, we found that maturation of oocytes enclosed by small antral follicles, isolated from nonprimed and PMSG-primed rats, could be induced by either FSH or forskolin but not by LH. cAMP determinations revealed that, in spite of the inability of LH to induce oocyte maturation, elevated concentrations of the nucleotide were detectable in small antral follicles exposed to this gonadotropin. Since granulosa cells isolated from the large but not the small antral follicles were stimulated by LH to generate cAMP, the elevation of cAMP concentrations in the small antral follicle apparently represented the response of the theca cells to this gonadotropin. Since it is the ability of the granulosa cells to interact with the hormone that determines whether or not oocyte maturation will occur, we suggest that the granulosa, but not the theca cells, mediate LH action to induce oocyte maturation.     

Towards a new understanding on the regulation of mammalian oocyte meiosis resumption

Mammalian oocytes reach prophase of first meiosis around the time of birth, and remain at this stage for months or years, depending on the species. Only after puberty will the fully-grown oocytes begin to resume meiosis which is stimulated by gonadotropin surge. It has long been known that a high level of intra-oocyte cyclic adenosine 3',5'-monophosphate (cAMP) prevents oocyte meiosis resumption as indicated by germinal vesicle breakdown (GVBD). Recently, guanosine triphosphate-binding (G) protein-coupled receptors/G proteins/adenyl cyclase pathway endogenous to the oocyte as well as cAMP diffusion from the somatic compartment through gap junctions have been implicated in maintaining cAMP at levels that prevent oocytes from resuming meiosis. Another second messager molecule, guanosine 3',5'-cyclic monophosphate (cGMP), has also recently been found to play important roles in maintaining oocyte meiosis arrest. cGMP in the follicular somatic cells diffuses into the oocyte and causes an increase in oocyte cAMP, presumably by acting on phosphodiesterase 3 (PDE3). The cGMP level in the somatic compartment of the follicle decreases in response to luteinizing hormone (LH), and this change may be mediated through the epidermal growth factor (EGF)-like factors and specific cGMP-phosphodiesterase subtype activity. It is well known that gonadotropic stimulation of meiotic resumption depends on mitogen-activated protein kinase (MAPK) activation in the somatic compartment of the follicle; recent studies show that LH, through cAMP/protein kinase A (PKA) and protein kinase C (PKC) pathways, induces the synthesis of paracine factors such as EGF-like facors and meiosis activating sterol (MAS) to regulate oocyte GVBD via the MAPK pathway in follicle cells. A recent granulosa cell-specific knockout study has for the first time provided in vivo evidence for the important role of extracellular regulated kinase 1 and 2 (ERK1/2), two main forms of MAPK, and their downstream molecules in granulosa cells in oocyte meiosis resumption. Unresolved questions and future directions on research regarding signaling changes in follicle cells and oocytes as well their communication in response to the gonadotropin surge are addressed in this review.     

Effects of high and low preovulatory concentrations of progesterone on ovulation from the isolated perfused rabbit ovary

Rabbit ovaries were isolated surgically before the ovulatory gonadotrophin stimulation and perfused in vitro. Untreated, control ovaries never ovulated. Ovaries treated in vitro with ovine LH ovulated 10-14 h later and the oocytes had undergone germinal vesicle breakdown (GVB). LH induced increases in progesterone secretion from the treated ovaries. A 3 beta-hydroxysteroid dehydrogenase blocker ('Compound A') effectively reduced progesterone secretion into the perfusate and follicular fluid to very low levels but had no effect on ovulation rate or on oocyte maturation. Excessively high progesterone levels were obtained artificially in perfusates by addition of exogenous steroid; the number of ovaries ovulating was markedly reduced but there was no effect on oocyte maturation. It is concluded that the rise in progesterone that normally occurs immediately after the LH surge is not a prerequisite for ovulation in the rabbit. However, progesterone may have a modifying effect on LH-induced follicle rupture when at a pharmacologically high level.     

Local roles of TGF-beta superfamily members in the control of ovarian follicle development

Members of the transforming growth factor-beta (TGF-beta) superfamily have wide-ranging influences on many tissue and organ systems including the ovary. Two recently discovered TGF-beta superfamily members, growth/differentiation factor-9 (GDF-9) and bone morphogenetic protein-15 (BMP-15; also designated as GDF-9B) are expressed in an oocyte-specific manner from a very early stage and play a key role in promoting follicle growth beyond the primary stage. Follicle growth to the small antral stage does not require gonadotrophins but appears to be driven by local autocrine/paracrine signals from both somatic cell types (granulosa and theca) and from the oocyte. TGF-beta superfamily members expressed by follicular cells and implicated in this phase of follicle development include TGF-beta, activin, GDF-9/9B and several BMPs. Acquisition of follicle-stimulating hormone (FSH) responsiveness is a pre-requisite for growth beyond the small antral stage and evidence indicates an autocrine role for granulosa-derived activin in promoting granulosa cell proliferation, FSH receptor expression and aromatase activity. Indeed, some of the effects of FSH on granulosa cells may be mediated by endogenous activin. At the same time, activin may act on theca cells to attenuate luteinizing hormone (LH)-dependent androgen production in small to medium-size antral follicles. Dominant follicle selection appears to depend on differential FSH sensitivity amongst a growing cohort of small antral follicles. Activin may contribute to this selection process by sensitizing those follicles with the highest "activin tone" to FSH. Production of inhibin, like oestradiol, increases in selected dominant follicles, in an FSH- and insulin-like growth factor-dependent manner and may exert a paracrine action on theca cells to upregulate LH-induced secretion of androgen, an essential requirement for further oestradiol secretion by the pre-ovulatory follicle. Like activin, BMP-4 and -7 (mostly from theca), and BMP-6 (mostly from oocyte), can enhance oestradiol and inhibin secretion by bovine granulosa cells while suppressing progesterone secretion; this suggests a functional role in delaying follicle luteinization and/or atresia. Follistatin, on the other hand, may favor luteinization and/or atresia by bio-neutralizing intrafollicular activin and BMPs. Activin receptors are expressed by the oocyte and activin may have a further intrafollicular role in the terminal stages of follicle differentiation to promote oocyte maturation and developmental competence. In a reciprocal manner, oocyte-derived GDF-9/9B may act on the surrounding cumulus granulosa cells to attenuate oestradiol output and promote progesterone and hyaluronic acid production, mucification and cumulus expansion.     

Ovary genes and molecular pathology

In the ovary, primordial follicles have to pass different stages in order to become preovulatory follicles. In the past few years, new genes and therefore new proteins have been recognized as major players in folliculogenesis. Atm, kit ligand and its receptor c-kit are necessary for the maintenance of ovarian follicle pool. GDF-9, BMP15, originating from the oocyte play a major role in early folliculogenesis. Pro and antiapoptotic proteins such as Bax and Bcl2 complete in granulosa cells, in order to maintain or not the follicle alive. FSH receptor is necessary for final follicular maturation, from the preantral stage and beyond. LH receptor is necessary for follicle ovulation. However, new genes and their regulation need to be identified as many ovarian diseases such as premature ovarian failure are not yet clarified.     

GPCR/EGFR cross talk is conserved in gonadal and adrenal steroidogenesis but is uniquely regulated by matrix metalloproteinases 2 and 9 in the ovary

Previous work has demonstrated that cross talk between G protein-coupled LH receptors and epidermal growth factor receptors (EGFR) is essential for LH-induced steroid production in ovarian follicles and testicular Leydig cells. Here we demonstrate that G protein-coupled receptor (GPCR)/EGFR cross talk is also required for ACTH-induced steroidogenesis in Y1 adrenal cells. Moreover, we confirm that the signaling pathway from GPCR to Erk activation is conserved in all three steroidogenic tissues. ACTH or LH induces Gα(s), resulting in elevated cAMP and protein kinase A activation. cAMP/protein kinase A then triggers EGFR trans-activation, which promotes Erk signaling and subsequent steroidogenesis. Interestingly, although EGFR trans-activation is conserved in all three tissues, the specific mechanisms regulating this receptor cross talk differ. ACTH and LH trigger matrix metalloproteinase (MMP)-mediated release of EGFR ligands in adrenal and gonadal cells, respectively. However, this extracellular, ligand-dependent EGFR transactivation is required only for LH-induced steroidogenesis in ovarian follicles, reflecting the unique requirement of cell-cell cross talk for ovarian steroid production. Furthermore, MMP2 and MMP9 appear to regulate LH-induced steroidogenesis in mouse ovarian follicles, because a specific MMP2/9 inhibitor as well as the MMP2/9 inhibitor doxycycline suppress LH-induced follicular steroid production in vitro. Notably, although EGFR or MMP inhibition minimally affects estrous cycling in female mice, they attenuate ovarian steroidogenesis in response to LHR overstimulation in vivo. These results may have implications with regard to EGFR inhibitor use in various cancers as well as in polycystic ovarian syndrome, where excess LH-driven ovarian androgen production might be controlled by MMP2/9 inhibition.     

Cyclic AMP changes in the component cells of Graafian follicles: possible influences on maturation in the follicle-enclosed oocytes of hamsters

Mature antral follicles were removed from the ovaries of pregnant mare serum gonadotropin (PMSG)-primed hamsters at proestrus prior to the LH surge. Following various incubation times with either LH (ovine) or FSH (rat), cAMP levels were determined in whole follicles, cumulus-oocyte complexes (COCs), and zona-intact or zona-free oocytes. LH produced a dose- and time-dependent change in follicle cAMP but had a minimal effect on the COCs and caused no change in cAMP in zona-free oocytes. By contrast, rFSH stimulated a small rise in follicular cAMP but significantly increased levels in COCs and zona-free oocytes. In a second series of experiments follicles were exposed for short periods to various additives after which they were washed and returned to hormone-free medium for a 6-hr total incubation period. LH (1 microgram/ml) initiated maturation in follicle-enclosed oocytes after a 5- to 15-min exposure period while groups incubated with 100 ng/ml required 60 min. FSH did not stimulate maturation after a 60-min exposure and when combined with 1 microgram or 100 ng/ml of LH negated the maturational effects seen with LH alone. It was postulated that the reason that lower concentrations of LH did not stimulate maturation following short-term incubations was due to an insufficient rise in cAMP. However, neither dbcAMP nor forskolin augmented the capacity of LH to initiate maturation following short-term exposure. By contrast dbcGMP and the guanylate cyclase activator, sodium nitroprusside (NP) did augment the maturation-inducing effects of LH. NP + LH raised cGMP concentrations in the follicle and oocyte and decreased follicular cAMP at 30 and 120 min. The results of this study indicate that the component cells within a follicle respond selectively with cAMP changes, depending on the gonadotropin, in a variable time- and dose-dependent manner. While LH is the more potent activator of cAMP in whole follicles, cAMP levels in the cumulus oophorus and oocyte show the greatest increase following exposure to FSH. LH was the more potent initiator of maturation, possibly through its effects on the mural granulosa cells. FSH appears to exert a more inhibitory role which may be due in part to elevated cAMP levels and/or a putitative inhibitor in the COC and oocyte.