This minireview summarizes the role that progesterone (P4) plays in regulating granulosa and luteal cell function. These actions include the stimulation of P4 synthesis and the inhibition of estrogen synthesis, mitosis, and apoptosis. P4 also plays a key role in the ovulatory process. Although P4′s actions are well documented, the mechanism or mechanisms that mediate all of these actions have not been defined. In addition to P4-induced gene transcription that is mediated by the nuclear P4 receptors (PGR-A and PGR-B), three other receptor/signal transduction pathways could account for P4′s intraovarian actions. These pathways could be mediated by 1) the PGR localizing at or near the plasma membrane and activating SRC family kinases, 2) a membrane progestin receptor that responds to P4 by lowering intracellular cAMP and increasing MAPK 3/1 activity, and 3) a membrane receptor complex composed of serpine 1 mRNA binding protein (also known as PAIRBP1 or RDA288) and progesterone receptor membrane component 1. Ligand activation of this complex likely leads to an increase in protein kinase G activity, the maintenance of low basal intracellular free calcium, and the inhibition of granulosa and luteal cell mitosis and apoptosis. Given the complexity of P4′s actions within the ovary, it is likely that all of these receptor/signal transduction pathways influence some aspect of ovarian function with the specific P4 response dependent on 1) the expression pattern of these putative P4 receptors, 2) the P4 binding affinity of each receptor system, and 3) the amount of available P4.

Recent research has focused on effects of the estrogen receptor acting at the level of the cell membrane in breast cancer. In this review we describe 17beta-estradiol (E2)-initiated membrane signaling pathways involving the activation of several kinases that contribute to the regulation of cell proliferation and prevention of apoptosis. Although classical concepts had assigned priority to the nuclear actions of estrogen receptor, recent studies document the additional importance of estrogen receptor residing in or near the plasma membrane. A small fraction of estrogen receptor is associated with the cell membrane and mediates the rapid effects of E2. Unlike classical growth factor receptors, such as insulin-like growth factor 1 receptor (IGF1R) and epidermal growth factor receptor (EGFR), estrogen receptor has no transmembrane and kinase domains and is known to initiate E2 rapid signals by forming a protein complex with many signaling molecules. The formation of the protein complex is a critical step, leading to the activation of the MAPK1/3 (also known as MAP kinase) and AKT1 (also known as Akt) pathways. A full understanding of the mechanisms underlying these relationships, with the ultimate aim of abrogating specific steps, should lead to more-targeted strategies for treatment of hormone dependent-breast cancer.

Epigenetic perturbations are assumed to be responsible for phenotypic abnormalities of fetuses and offspring originating from in vitro embryo techniques. We studied 29 viable Day-80 bovine fetuses to assess the effects of two in vitro fertilization protocols (IVF1 and IVF2) on fetal phenotype and genomic cytosine methylation levels in liver, skeletal muscle, and brain. The IVF1 protocol employed 0.01 U/ml of FSH and LH in oocyte maturation medium and 5% estrous cow serum (ECS) in embryo culture medium, whereas the IVF2 protocol employed 0.2 U/ml of FSH and no LH for oocyte maturation and 10% ECS for embryo culture. Comparisons with in vivo–fertilized controls (n = 14) indicated an apparently normal phenotype for IVF1 fetuses (n = 5), but IVF2 fetuses (n = 10) were significantly heavier (19.9%) and longer (4.7%), with increased heart (25.2%) and liver (27.9%) weights, and thus displayed an overgrowth phenotype. A clinicochemical screen of 18 plasma parameters revealed significantly increased levels of insulin-like growth factor 1 (40.8%) and creatinine (37.5%) in IVF2, but not in IVF1, fetuses. Quantification of genomic 5-methylcytosine (5mC) by capillary electrophoresis indicated that both IVF1 and IVF2 fetuses differed from controls. We observed significant DNA hypomethylation in liver and muscle of IVF1 fetuses (−16.1% and −9.3%, respectively) and significant hypermethylation in liver of IVF2 fetuses ( 11.2%). The 5mC level of cerebral DNA was not affected by IVF protocol. Our data indicate that bovine IVF procedures can affect fetal genomic 5mC levels in a protocol- and tissue-specific manner and show that hepatic hypermethylation is associated with fetal overgrowth and its correlated endocrine changes.

A need exists for broadly applicable biomarkers of pregnancy outcome in population-based studies that assess environmental hazards to human reproduction. Previous studies have demonstrated that during the periimplantation period, measures of the circulating levels of immunoreactive hCG (IhCG) are not predictive of pregnancy outcome, whereas measurements of the circulating levels of bioactive hCG (BhCG) provide information relating to pregnancy outcome and might provide the basis for an early biomarker of pregnancy outcome. However, for this biomarker to have broad application in population-based studies, it must be adapted to urinary hCG metabolites. The principle objective of the present study was to characterize the periimplantation excretion patterns of urinary hCG metabolites of pregnancies that resulted in live birth (LB), early pregnancy loss (EPL), and recognized clinical abortion (CAB) with an immunoenzymometric assay specific to intact hCG and an LH/chorionic gonadotropin cellular bioassay as the basis for a preliminary comparison between successful (LB) and failing (EPL and CAB) outcome groups. Automated immunoassays for FSH and hCG were used to define each conceptive cycle's implantation window. The timing of first hCG detection was significantly later for the EPL group. Pregnancies that resulted in LB had consistently rising average daily IhCG and BhCG levels, with no significant differences when average daily IhCG and BhCG measurements were compared (Student t-test, P > 0.05), whereas pregnancies that resulted in CAB and EFL had lower average daily IhCG and BhCG levels that increased inconsistently. These findings demonstrate that critical information related to pregnancy outcome may be present when multiple urinary hCG isoforms are measured. Further data suggest that the rate of change for the ratio of daily BhCG over IhCG levels might be useful as the basis of a broadly applicable early biomarker for pregnancy outcome.

Body growth during critical periods is known to be an important factor in determining the age of maturity and fecundity in fish. However, the endocrine mechanisms controlling oogenesis in fish and the effects of growth on this process are poorly understood. In this study interactions between the growth and reproductive systems were examined by monitoring changes in various components of the FSH-ovary axis, plasma insulin-like growth factor 1 (Igf1), and ovarian gene expression in relation to body and previtellogenic oocyte growth in coho salmon. Samples were collected from females during two hypothesized critical periods when growth influences maturation in this species. Body growth during the fall-spring months was strongly related to the degree of oocyte development, with larger fish possessing more advanced oocytes than smaller, slower growing fish. The accumulation of cortical alveoli in the oocytes was associated with increases in plasma and pituitary FSH, plasma estradiol-17beta, and ovarian steroidogenic acute regulatory protein (star) gene expression, whereas ovarian transcripts for growth hormone receptor and somatolactin receptor decreased. As oocytes accumulated lipid droplets, a general increase occurred in plasma Igf1 and components of the FSH-ovary axis, including plasma FSH, estradiol-17beta, and ovarian mRNAs for gonadotropin receptors, star, igf1, and igf2. A consistent positive relationship between plasma Igf1, estradiol-17beta, and pituitary FSH during growth in the spring suggests that these factors are important links in the mechanism by which body growth influences the rate of oocyte development.

In this study, we discovered that embryos sense shear stress and sought to characterize the kinetics and the enzymatic mechanisms underlying induction of embryonic lethality by shear stress. Using a rotating wall vessel programmed to produce 1.2 dynes/cm² shear stress, it was found that shear stress caused lethality within 12 h for E3.5 blastocysts. Embryos developed an approximate 100% increase in mitogen-activated protein kinase 8/9 (formerly known as stress-activated protein kinase/junC kinase 1/2) phosphorylation by 6 h of shear stress that further increased to approximately 350% by 12 h. Terminal deoxynucleotidyltransferase dUTP nick end labeling/apoptosis was at baseline levels at 6 h and increased to approximately 500% of baseline at 12 h, when irreversible commitment to death occurred. A mitogen-activated protein kinase 8/9 phosphorylation inhibitor, D-JNKI1, was able to inhibit over 50% of the apoptosis, suggesting a causal role for mitogen-activated protein kinase 8/9 phosphorylation in the shear stress-induced lethality. The E2.5 (compacted eight-cell/early morula stage) embryo was more sensitive to shear stress than the E3.5 (early blastocyst stage) embryo. Additionally, zona pellucida removal significantly accelerated shear stress-induced lethality while having no lethal effect on embryos in the static control. In conclusion, preimplantation embryos sense shear stress, chronic shear stress is lethal, and the zona pellucida lessens the lethal and sublethal effects of shear stress. Embryos in vivo would not experience as high a sustained velocity or shear stress as induced experimentally here. Lower shear stresses might induce sufficient mitogen-activated protein kinase 8/9 phosphorylation that would slow growth or cause premature differentiation if the zona pellucida were not intact.

Morphological male sex determination is dependent on migration of endothelial and preperitubular cells from the adjacent mesonephros into the developing testis. Our hypothesis is that VEGFA and its receptor KDR are necessary for both testicular cord formation and neovascularization. The Vegfa gene has 8 exons with many splice variants. Vegfa120, Vegfa164, and Vegfa188 mRNA isoforms were detected on Embryonic Day (E) 13.5 (plug date = E0) in the rat. Vegfa120, Vegfa144, Vegfa164, Vegfa188, and Vegfa205 mRNA were detected at E18 and Postnatal Day 3 (P3). Kdr mRNA was present on E13.5, whereas Fms-like tyrosine kinase 1 receptor (Flt1) mRNA was not detected until E18. VEGFA protein was localized to Sertoli cells at cord formation and KDR to germ and interstitial cells. The VEGFA signaling inhibitors SU1498 (40 μM) and VEGFR-TKI (8 μM) inhibited cord formation in E13 testis cultures with 90% reduced vascular density (P < 0.01) in VEGFR-TKI-treated organs. Furthermore, Je-11 (10 μM), an antagonist to VEGFA, also perturbed cord formation and inhibited vascular density by more than 50% (P < 0.01). To determine signal transduction pathways involved in VEGFA's regulation of testis morphogenesis, E13 testis were treated with LY 294002 (15 μM), a phosphoinositide 3-kinase (PI3K) pathway inhibitor, resulting in inhibition of both vascular density (46%) and cord formation. Thus, we support our hypothesis and conclude that VEGFA, secreted by the Sertoli cell, is involved in both neovascularization and cord formation and potentially acts through the PI3K pathway during testis morphogenesis to elicit its effects.

Spermatogenesis originates from a small number of spermatogonial stem cells that can reinitiate spermatogenesis and produce germ cell colonies following transplantation into infertile recipient testes. Although several previous studies have suggested a single-cell origin of germ cell colonies, only indirect evidence has been presented. In this investigation, we tested the clonal origin hypothesis using a retrovirus, which could specifically mark an individual spermatogonial stem cell. Spermatogonial stem cells were infected in vitro with an enhanced green fluorescence protein-expressing retrovirus and subsequently transplanted into infertile recipient mice. Live haploid germ cells were recovered from individual colonies and were microinjected into eggs to create offspring. In total, 45 offspring were produced from five colonies, and 23 (51%) of the offspring were transgenic. Southern blot analysis indicated that the transgenic offspring from the single colony carried a common integration site, and the integration site was different among the transgenic offspring from different colonies. These results provide evidence that germ cell colonies develop from single spermatogonial stem cells.

Wnt genes regulate a diverse range of developmental processes, including placental formation. Activation of the WNT pathway results in translocation of beta-catenin (CTNNB1) into the nucleus and the subsequent activation of transcription factors that promote proliferation. The secreted frizzled related proteins (SFRPs) are thought to inhibit WNT signaling by binding to the WNT ligand or its frizzled receptor. In this study, we compared the expression patterns of one of these secreted molecules, SFRP4, in the two morphologically and functionally distinct regions of the rat placenta during the last third of pregnancy. In addition, we assessed whether placental SFRP4 expression is altered in a model of glucocorticoid-induced placental growth restriction. Temporal analyses of the rat placenta by quantitative RT-PCR, in situ hybridization, and immunohistochemistry during the final third of pregnancy demonstrated elevated levels of Sfrp4 mRNA and SFRP4 protein near term, specifically in trophoblast cells of the basal zone. This increase in expression of SFRP4 in basal zone trophoblasts was associated with a reduction in CTNNB1 nuclear translocation, consistent with inhibition of the WNT pathway. Maternal dexamethasone treatment (1 μg/ml of drinking water, Days 13–22), which has previously been shown to reduce placental growth, further increased the expression of Sfrp4 mRNA in both the basal and labyrinth zones of the placenta at Day 22. Collectively, these data demonstrate that increased expression of SFRP4 is associated with reduced growth of placental regions in normal pregnancy and after glucocorticoid-induced growth retardation. These observations, together with associated changes in CTNNB1 localization, support the hypothesis that increased placental expression of SFRP4 inhibits the WNT pathway and thereby influences placental growth via effects on cell fate signaling.

N-arachidonoylethanolamide (anandamide [AEA]) is the main endocannabinoid described to date in the testis. It exerts its effects through the activation of G-protein coupled cannabinoid receptors (CNR). However, the activity of AEA in controlling male reproduction is still poorly known. Here we provide direct evidence on the presence of the “endocannabinoid system,” constituted by type-1 cannabinoid receptor (CNR1) and fatty acid amide hydrolase (FAAH), in the frog Rana esculenta testis demonstrating its expression in tubular compartment. In fact, during the annual reproductive cycle, both proteins increase in September, when the appearance of spermatids (SPT) occurs. Immunocytochemistry confirms their localization in germ cells and, in particular, in elongated SPT. Signals are still present in spermatozoa (SPZ), as demonstrated by Western blot analysis. Furthermore, the activation of CNR1 reduces sperm motility. Comparative research, carried out using mouse and rat SPZ, definitely indicates that the endocannabinoid system operates in SPZ of phylogenetically distant species. A conserved physiological role of endocannabinoid system in controlling the inhibition of sperm motility is suggested.

During human pregnancy, the trophoblast layer is in direct contact with maternal albumin. In contrast to immunoglobulins, albumin does not cross the placental barrier. However, albumin affects the trophoblast placental lactogen and chorionic gonadotroph secretion. The present study investigated the interaction between albumin and syncytiotrophoblast using human term placental explants. Bovine serum albumin, labeled with either ¹²⁵I or fluorescein isothio-cyanate, was taken up rapidly by placental explants. This process was temperature-sensitive. The internalized labeled BSA quickly outflowed from the tissue at the maternal side, largely without any major modification in molecular weight. Colchicine (1 mM), which disrupts the microtubule network, or cytochalasin B (40 μM), which disassembles filamentous actin, did not interfere with the placental transmembrane movements of labeled BSA. Megalin, clathrin, and caveolin 1 are three membrane proteins associated with albumin endocytosis in other tissues, but only megalin and clathrin were detected in the syncytiotrophoblast layer by immunohistochemistry. The uptake of labeled BSA into placental explants was not modified by 4,4′-diisothiocyanatostilbene-2,2′-disulfonic acid (1 mM) or 5-nitro-2-(3-phenylpropylamino)benzoic acid (100 μM), two pharmacological tools known to disturb megalin-mediated albumin endocytosis. By contrast, methyl-β-cyclodextrin (10 mM) and chlorpromazine (1.4 mM), both of which disrupt the clathrin-mediated endocytotic system, significantly reduced the uptake of labeled BSA. These data suggest, to our knowledge for the first time, that maternal albumin is actively internalized into the human trophoblast according to an apical recycling pathway. This temperature-sensitive process does not depend on an intact cytoskeleton, but it is associated with a clathrin-mediated endocytotic system.

FSH receptor has been shown to be specifically expressed only in the Sertoli cells in males. In one of our studies that consisted of deprival of endogenous FSH in immature rats and adult bonnet monkeys, atrophy of the epididymis was observed, cauda region being the most affected. Although epididymis is an androgen-dependent tissue, the changes in histology of the cauda region were observed without any associated change in the levels of testosterone in FSH-deprived animals. Considering this, it was of interest to evaluate the possibility of epididymis being a direct target for FSH action. In the present study, we have examined the expression of FSH receptor in the epididymis of rat and monkey. In the cauda region of rat epididymis, FSH receptor expression was demonstrated by RT-PCR and Northern and Western blot analyses. FSH receptor was found to be functional as observed by its ability to bind ¹²⁵IoFSH, by an increase in cAMP production, and by BrdU incorporation following addition of FSH under in vitro conditions. These results suggest the possibility of a role for FSH in regulating the growth of the epididymis.

Endometriosis is characterized by ectopic growth of endometrial tissues. Statins, inhibitors of 3-hydroxy-3methylglutaryl-coenzyme A reductase (HMGCR), have been shown to decrease proliferation of several mesenchymal tissues. Actions of statins may be related to decreased availability of cholesterol as well as intermediate metabolites of the mevalonate pathway downstream of HMGCR. This study was designed to evaluate effects of statins on growth of endometrial stromal cells and to investigate mechanisms of these effects. Human endometrial stromal cells were cultured in the absence and in the presence of serum and with or without mevastatin and simvastatin. DNA synthesis and viable cell numbers were determined. Effects of statins were also evaluated in the presence of mevalonate and squalene. Furthermore, effects on phosphorylation of mitogen-activated protein kinase 3/1 (MAPK3/1) (also known as extracellular signal-regulated kinase [ERK1/2]) were determined. Mevastatin and simvastatin induced a concentration-dependent inhibition of DNA synthesis and viable cell count in chemically defined media and in the presence of serum. Mevalonate, but not squalene, abrogated inhibitory effects of statins on cell proliferation. Statins inhibited MAPK3/1 phosphorylation. This is the first study demonstrating that statins inhibit growth of endometrial stromal cells. This effect is also demonstrable in the presence of a supply of cholesterol and may be related to decreased activation of MAPK3/1. The present observations may be relevant to potential therapeutic use of statins in conditions such as endometriosis.

The human placenta is a unique organ in terms of oxygenation as it undergoes a transition from a low to a more oxygenated environment. This physiological switch in oxygen tension is a prerequisite for proper placental development and involves the hypoxia inducible factor (HIF). HIF is stable and initiates gene transcription under hypoxia, whereas in normoxia, interaction with the von Hippel-Lindau tumor suppressor protein (VHL) leads to rapid degradation of the HIF1A subunit. The degradation requires formation of a multiprotein complex (VHL^CBC) and hydroxylation of HIF1A proline residues via members of the egg-laying-defective nine (EGLN) family. Herein, we have investigated the regulatory mechanisms of HIF1A expression during human placental development. Expression of HIF1A and VHL was high at 7–9 wk of gestation, when oxygen tension is low, and decreased when placental oxygen tension increases (10–12 wk of gestation). During early placentation, HIF1A localized in cytotrophoblasts, while VHL was present in syncytiotrophoblasts. At 10–12 wk, VHL appeared in cytotrophoblast cells, which coincided with the disappearance of HIF1A. At the same time the association of VHL and Cullin 2 as well as ubiquitination of HIF1A was maximal. EGLN1, EGLN2, and EGLN3 were also temporally expressed in an oxygen-dependent fashion, with greatest mRNA expression at 10–12 wk of gestation. Inhibition of EGLN activity increased HIF1A stability in villous explants and stimulated transforming growth factor beta 3 (TGFB3) expression consistent with promoter analyses showing that HIF1A transactivates TGFB3. These data demonstrate that during placental development, HIF1A is regulated by temporal and spatial changes in expression and association of molecules forming the multi-protein VHL^CBC complex as well as prolyl hydroxylase activities.

Somatic nuclear transfer (NT) in cattle is often complicated by fetal oversize (i.e., large offspring syndrome), hydrallantois, and placentomegaly in late gestation. The aims of this work were to obtain data on the placentome structure in NT-recipient cows with hydrallantois (NTH) and to relate these with fetal and placental weights to better understand the abnormalities observed in NTH pregnancies during the third trimester. Pregnant cows were slaughtered between Gestation Days 180 and 280. The fetuses were weighed, and the placentomes were numbered and weighed. Placentomes were examined by histologic and stereological techniques. Macroscopic data showed that placental overgrowth preceded fetal overgrowth, and the ratio of the fetal to the total placentome weight in the NTH group was lower than that in controls after Gestation Day 220. This suggests that placental overgrowth is due to placental default rather than due to fetal overgrowth, as shown also by stereological analysis showing primary deregulation of the growth of cotyledonary tissues. Observed alterations, such as thinning of the maternal epithelium within placentomes and increased trophoblastic surface, could be secondary adaptations. Thus, placental growth deregulations would be due to modifications of the expression of placental factors. Various examples of placental deficiency were observed, suggesting that some fetal abnormalities observed in NTH calves, such as enlarged heart, enlarged umbilical cord, and abdominal ascites, are consequences of placental dysfunction. Therefore, the condition described by the term “large offspring syndrome” might better be described by “large placenta syndrome,” because this syndrome affects an average of 50% of late-gestation NT pregnancies. No conclusion can be drawn from this work on apparently normal pregnancies.

The objective of the present study was to examine the expression of Toll-like receptors (TLRs) by mouse uterine epithelial cells and to determine if stimulation of the expressed TLR induces changes in cytokine and/or chemokine secretion. Using RT-PCR, the expression of TLRs 1–6 by mouse uterine epithelial cells was demonstrated, with TLRs 7–9 expressed only periodically. In the absence of pathogen-associated molecular patterns, polarized uterine epithelial cells constitutively secrete interleukin (IL) 1A, cysteine-cysteine ligand (CCL) 2, IL6, granulocyte-macrophage colony-stimulating factor 2 (CSF2), tumor necrosis factor A (TNFA), CSF3, and IL8 in vitro, with levels of cytokines/chemokines secreted into the apical compartment being significantly greater than those released into the basolateral compartment. When added to the apical surface for 48 h before analysis, the TLR2-agonist Pam₃Cys-Ser-(Lys)₄ and TLR1/6-agonist peptidoglycan increased epithelial cell apical secretion of IL1A, CCL2, and IL6 and apical/basolateral bidirectional secretion of CSF2, TNFA, CSF3, and IL8 when compared to controls. The TLR3-agonist poly (I:C) significantly increased bidirectional secretion of CCL2, IL6, TNFA, and CSF2 and basolateral secretion of CSF3. Lastly, the TLR4-agonist lipopolysaccharide increased bidirectional secretion CCL2, CSF2, TNFA, CSF3, and IL8 and apical secretion of IL6. These results indicate that mRNAs for Tlr1 through Tlr6 are expressed by uterine epithelial cells and that treatment with specific TLR agonists alters the expression of key chemokines and proinflammatory cytokines that contribute to the defense of the uterus against potential pathogens.

EGF or TGFB1 alone stimulates but together attenuate granulosa cell DNA synthesis. Intact preantral follicles from hamsters were cultured with TGFB1, EGF, or both to reveal the mechanisms of such unique regulation. Follicular CCND2 (also known as cyclin D2), CDKN1B (also known as p27^kip1), and the involvement of appropriate signaling intermediaries and kinases were examined. TGFB1, acting via SMAD2 and SMAD3, antagonized the degradation of CCND2 protein by blocking its phosphorylation. In contrast, TGFB1 supported CDKN1B degradation by involving MAPK14 (also known as p38 Map Kinase) and PKC, resulting in CDK4 activation and DNA synthesis. EGF via MAPK3/1 maintained functional levels of CCND2 through CCND2 synthesis as well as degradation. EGF and TGFB1 together inhibited CDK4 activation and DNA synthesis. EGF attenuated TGFB1 stimulated phosphorylation of SMAD3, TGFB1-induced activation of MAPK14 and PKC, and TGFB1 suppression of CCND2 degradation. In contrast, TGFB1 suppressed EGF-induced increase in CCND2 mRNA levels. The final outcome was CCND2 degradation without replenishment and decreased activities of MAPK14 and PKC leading to suppression of CDK4 activation. The results indicate that each growth factor involves a separate mechanism to maintain an effective level of CCND2 in granulosa cells for the activation of CDK4 and induction of DNA synthesis. However, their simultaneous action is inhibitory to follicular DNA synthesis because they counteract each other's activity by interfering at specific sites. Because both EGF and TGFB1 are present in granulosa cells, this mechanism may explain how their effects are temporally modulated for granulosa cell proliferation and folliculogenesis.

The objective was to reveal whether a protein kinase C (PKC [all isozymes])-mediated self-sustaining MAPK3/1 (3/1 extracellular signal regulated kinase 2/1, also known as ERK2/1) activation loop was necessary for FSH- or epidermal growth factor (EGF)-induced DNA synthesis in the granulosa cells of intact preantral follicles. For this purpose, hamster preantral follicles were cultured with FSH or EGF in the presence of selective kinase inhibitors FSH or EGF phosphorylated RAF1, MAP2K1, and MAPK3/1. However, a relatively higher dose of EGF was necessary to sustain the MAPK3/1 activity, which was essential for cyclin-dependent kinase 4 (CDK4) activation and DNA synthesis. In intact preantral follicles, FSH or EGF stimulated DNA synthesis only in the granulosa cells. Sustained activation of MAPK3/1 beyond 3 h was independent of EGFR kinase activity but dependent on PKC activity, which appeared to form a self-sustaining MAPK3/1 activation loop by activating RAF1, MAP2K1, and PLA2G4 (phospholipase A2 [all cytosolic isozymes]). Inhibition of PKC activity as late as 4 h after the administration of FSH or EGF arrested DNA synthesis, which corresponded with attenuated phosphorylation of RAF1 and MAPK3/1, thus suggesting an essential role of PKC in MAPK3/1 activation. Collectively, these data present a novel self-sustaining mechanism comprised of MAPK3/1, PLA2G4, PKC, and RAF1 for CDK4 activation leading to DNA synthesis in granulosa cells. Either FSH or EGF can activate the loop to activate CDK4 and initiate DNA synthesis; however, consistent with our previous findings, FSH effect seems to be mediated by EGF, which initiates the event by stimulating EGFR kinase.