The expression of genes involved in luteinization (Star, Cyp11a1, and Hsd3b1) of granulosa cells and cumulus expansion (Has2) of cumulus cells was induced by hCG injection (Fig 3D). When granulosa cells were cultured on uncoated wells, the morphology of granulosa cells was circular and the cell membrane was lined with actin polymers (Fig 4A). If the response to FSH is excessive, the trigger mechanism (hCG injection) can be replaced by using a single injection GnRH agonist, which elicits a short LH surge—sufficient to mature the eggs, but the degree of subsequent luteinization is diminished, and the risk of OHSS is almost eliminated. Ovulated oocytes were collected from oviducts and counted at 16 h after hCG injection. Stress fibers consist of actin and myosin, and actin polymerization is induced in a Rho GTPase family-dependent manner [30][31][32]. No, Is the Subject Area "Ovulation" applicable to this article? FBS was added to this culture medium because FBS-containing inter α trypsin inhibitor has been known to be essential for cumulus expansion. A major determinant of normal corpus luteum formation and function is optimal development of the corpus luteum predecessor, the dominant follicle. Transcriptional regulation is a dynamic process that has for the most part been studied in a static context. Values are the mean ± SEM of five mice. Huet et al. Many clinics use induction of menses routinely to ‘program’ cycles and control the number of egg collections in any one week. *; Y15 treatment significantly decreased the progesterone level when granulosa cells were cultured on fibronectin-coated wells (p<0.05). ERK1/2-null mice do not exhibit Cyp19a1 downregulation in response to an ovulatory concentration of hCG369 (Figure 20.13). GnRH antagonists prevent premature luteinization without the pre-treatment required with GnRH agonists. Granulosa cells were harvested by needle puncture from immature mice treated with eCG at 23 days of age. Phosphorylation of Tyr397 was not induced by fibronectin-coated plate and by additional AREG to non-coded plate (Fig 2B). The best known are effected by epigenetic mechanisms modification of histones, particularly histones H3 and H4. 1994 Jul;72(7):1857-72. doi: 10.2527/1994.7271857x. Three mice were used for sampling granulosa cells in each treatment group at each time point. Values are the mean ± SEM of fifteen granulosa cells. The cell membrane was lined with actin polymers and spots of actin polymers were also observed in the cytoplasm of granulosa cells cultured on the serum-coated plate (Fig 4A). To clear this hypothesis, the localizations of fibronectin and its receptor integrin were observed by immunofluorescence technique. After washing with 0.3% (v/v) Triton X-100 in PBS, the staining was visualized with Cy3-conjugated goat anti-rabbit IgG (1:200) (Sigma) and FITC-conjugated goat anti-rabbit IgG (1:200) with counterstaining by 4,6-diamidino-2-phenylindole (DAPI, Vector Laboratories Inc). https://doi.org/10.1371/journal.pone.0192458.s004, https://doi.org/10.1371/journal.pone.0192458.s005, https://doi.org/10.1371/journal.pone.0192458.s006. Because it has been known that serum contains fibronecin, granulosa cells were cultured in fetal bovine serum (FBS, cat#10438–018, Gibco)-coated or uncoated glass bottom dishes in serum-free medium (DMEM:F12 containing penicillin-streptomycin) with 100 ng/ml AREG and 3 μg/ml fibronectin for 12 h. Ovaries of immature mice primed with eCG for 48 h contain multiple preovulatory follicles. Dulbecco’s modified Eagle’s medium (DMEM)-F12 medium and penicillin-streptomycin from Invitrogen (Carlsbad, CA, USA); oligonucleotide poly-(dT) from Invitrogen, AMV reverse transcriptase from Promega (Madison, WI, USA). Repression of inhibin expression during the peri-ovulatory time window permits the secondary surge of FSH on the morning of estrous that contributes to follicle recruitment.508 Reduced Inha transcriptional activity is initially accomplished by the rapid, albeit transient, induction in mural granulosa cells of the immediate early gene Crem (cAMP response element modulator) that encodes the inducible cAMP early repressor ICER.346 ICER, along with CREB, C/EBPβ, and NFIL3 are basic leucine zipper transcription factors that bind DNA and homo- or heterodimerize to activate or repress transcription. Competing interests: The authors have declared that no competing interests exist. Non-expanded COCs were selected and were cultured in the medium containing 1% (v/v) of FBS with 100 ng/ml AREG and/or 100 nM FAK inhibitor (Y15) in the presence of 4 mM of hypoxanthine for 16 h. Red signal is F-actin, green signal is α/β Tubulin and blue signal is DAPI. here. Formal analysis, Dynamics of Myc/Max/Mad expression during luteinization of primate granulosa cells in vitro: association with periovulatory proliferation. In the mutant mouse, almost all genes reported to be expressed in granulosa cells and cumulus cells during ovulation process are not expressed after human chorionic gonadotropin (hCG) injection [4], indicating that the function of EGFR changes the gene expression pattern from the follicular development stage to ovulation process. Cell models of luteinization, along with some of the conflicting observations on the luteinization process, are discussed in this review. Values are the mean ± SEM of three replicates. Proliferation ceases, the structure formed following ovulation becomes highly vascularized, and luteal cells produce high levels of progesterone. The Stemness of Human Ovarian Granulosa Cells and the Role of Resveratrol in the Differentiation of MSCs-A Review Based on Cellular and Molecular Knowledge. The requirement of ERK1/2 activation for Lhcgr mRNA downregulation is supported by results with ERK1/2-null mice that fail to show a decline in Lhcgr mRNA post hCG.369 The relevant ERK1/2 target has not been identified, but might be LRBP itself, especially since LRBP is readily detectable in granulosa cell extracts in the absence of hCG, consistent with regulation by a posttranslational mechanism.524 While the mechanism by which surface LH receptors are downregulated in granulosa cells and corpora lutea following a bolus of LH or hCG has been clearly defined, the physiological consequence of blocking this response, especially in granulosa cells, has not to our knowledge been investigated. Finally, using a pharmacological approach, the roles of FAK and the binding of fibronectin to integrins in granulosa cells and cumulus cells during ovulation were analyzed in vivo and in vitro. Interestingly, hyperstimulation syndrome rarely occurs after a spontaneous LH surge induced by increasing estrogen levels. eCollection 2019. C/EBPα/β-null mice exhibit the same phenotype,495 consistent with evidence that expression and activation of C/EBPβ is dependent on ERK1/2 activation369 and with evidence of the repressive actions of C/EBPβ.510 Likewise, granulosa cells expressing EGF receptors with compromised receptor tyrosine kinase activity exhibit decreased C/EBPβ and fail to downregulate Cyp19a1492 (see Figure 20.13). Except where noted, content and user contributions on this site are licensed under CC BY-SA 4.0 with attribution required. It has recently been reported that histone H3 phosphorylation is induced by FSH [115] and LH [116] in rat granulosa cells. The relative aromatizing ability of bovine luteinizing granulosa cells and dispersed luteal cells in tissue culture was studied. Values are mean +/- SEM of 3 replicates. Steroidogenic, Metabolic, and Immunological Markers in Dairy Cows Diagnosed With Cystic Ovarian Follicles at Early and Mid-Late Lactation. 2019 Sep 26;6:324. doi: 10.3389/fvets.2019.00324. Values are the mean ± SEM of three replicates. EGF-like factor acts on granulosa cells and cumulus cells enclosed by fibronectin to drive the dynamic changes of the cytoskeleton with the differentiation of each cell type, cumulus expansion of cumulus cells, and luteinization of granulosa cells. Some COCs were treated with AREG and 100 nM Y15 or 100 μM RGD peptide (RGD; arginine-glycine-aspartate, tripeptide). (A): Levels of FAK (total FAK), phosphorylated forms of FAK (Tyr397 and Tyr925), and β-actin in whole ovary samples were detected by western blot analyses. Endocrinology. We investigated the kinetic changes of fibronectin and integrin in periovulatory follicles and their roles in activation of the FAK pathway. (A): FAK inhibitor Y15 was injected into three immature mice with hCG and the ovary was collected at 4 h after hCG injection. (B): Effect of Y15 on cumulus expansion (surface area of COCs in periovulatory follicles of the ovary). ScienceDirect ® is a registered trademark of Elsevier B.V. ScienceDirect ® is a registered trademark of Elsevier B.V. URL: https://www.sciencedirect.com/science/article/pii/B9780124445628500124, URL: https://www.sciencedirect.com/science/article/pii/B9780128042144000026, URL: https://www.sciencedirect.com/science/article/pii/B978012397175300020X, URL: https://www.sciencedirect.com/science/article/pii/B978145574858700024X, URL: https://www.sciencedirect.com/science/article/pii/B9780323032476100024, URL: https://www.sciencedirect.com/science/article/pii/B9780080970370000609, URL: https://www.sciencedirect.com/science/article/pii/B9780323189071001256, URL: https://www.sciencedirect.com/science/article/pii/B9780128007495000262, URL: https://www.sciencedirect.com/science/article/pii/B9780323479127000081, Encyclopedia of Reproduction (Second Edition), 2018, Nicolas M. Orsi, ... Michele Cummings, in, Knobil and Neill's Physiology of Reproduction (Fourth Edition), Robert L. Rosenfield MD, ... Sally Radovick MD, in, In Vitro Fertilization and Embryo Transfer (IVF-ET), The Immunoassay Handbook (Fourth Edition), Endocrinology: Adult and Pediatric (Seventh Edition), Human Chorionic Gonadotropin (HGC) (Second Edition), Ovarian hyperstimulation syndrome occurs after, Yen and Jaffe's Reproductive Endocrinology (Eighth Edition). Oxytocin is expressed in the CL of cows, primates, and pigs [65, 66]. Conversely, histone modifications linked to repressed genes, H3K27me3 and H3K9me3, are low in preovulatory cells and substantially increase in ovulatory cells and further increase in luteal cells obtained 6 days post hCG.512 DNA methylation within the cAMP response element, as determined by bisulfite sequencing, is low in preovulatory and ovulatory cells and markedly enhanced in corpora lutea.

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