排卵過程における黄体化制御および卵成熟に関与するfibronectinの機能解析

内容の要約広島大学(Hiroshima University)博士(農学)Doctor of Agriculturedoctora

The acceleration of reproductive aging in Nrg1flox/flox;Cyp19-Cre female mice

Irregular menstrual cycles, reduced responses to exogenous hormonal treatments, and altered endocrine profiles (high FSH/high LH/low AMH) are observed in women with increasing age before menopause. In this study, because the granulosa cell-specific Nrg1 knockout mice (gcNrg1KO) presented ovarian and endocrine phenotypes similar to older women, we sought to understand the mechanisms of ovarian aging and to develop anewstrategy for improving fertility in older women prior to menopause. In the ovary of 6-month-old gcNrg1KO mice, follicular development was blocked in bilayer secondary follicles and heterogeneous cells accumulated in ovarian stroma. The heterogeneous cells in ovarian stroma were distinguished as two different types: (i) the LH receptor-positive endocrine cells and (ii) actin-rich fibrotic cells expressing collagen. Both the endocrine and fibrotic cells disappeared following long-term treatment with a GnRH antagonist, indicating that the high levels of serum LH induced the survival of both cell types and the abnormal endocrine profile to reduce fertility. Moreover, follicular development to the antral stages was observed with reduced LH and the disappearance of the abnormal stromal cells. Mice treated with the GnRH antagonist regained normal, recurrent estrous cycles and continuously delivered pups for at least for 3 months. We conclude that endocrine and matrix alternations occur within the ovarian stroma with increasing age and that abolishing these alternations resets the cyclical release of LH. Thus, GnRH antagonist treatments might provide a new, noninvasive strategy for improving fertility in a subset of aging women before menopause.This work was supported in part by The Japan Society for the Promotion of Science (JSPS) KAKENHI, JP24688028, JP 16H05017 (to MS) and JP15J05331 (to TU), by Japan Agency for Medical Research and Development (AMED) 16gk0110015 h0001 (to MS), and by National Institute of Health (NIH)-HD-076980 (to JSR)

Inductions of granulosa cell luteinization and cumulus expansion are dependent on the fibronectin-integrin pathway during ovulation process in mice

<div><p>It has been known that EGF-like factor secreted from LH-stimulated granuloma cells acts on granulosa cells and cumulus cells to induce ovulation process. Granulosa cells are changed the morphology with differentiating cell functions to produce progesterone. Cumulus cells are detached to make a space between the cells to accumulate hyaluronan rich matrix. LH also changes extracellular matrix (ECM) components including fibronectin in the follicular walls and granulosa cell layers. EGF like factor and fibronectin synergistically play important roles in numerous cell functions, especially cancer cell migration, estimating that fibronectin would impact on granulosa cells and cumulus cells. To clear this hypothesis, the localizations of fibronectin and its receptor integrin were observed by immunofluorescence technique. The functions were monitored by the detection of downstream signaling pathway, focal adhesion kinase (FAK). The pharmacological approach in both <i>in vivo</i> and <i>in vitro</i> were used for analyzing the physiological roles of FAK during ovulation process. The immunofluorescence staining revealed that fibronectin and integrin were observed in granulosa cells, cumulus cells and the space between cumulus cells and oocyte at 4 and 8 h after hCG injection. Concomitantly with the changes of fibronectin-integrin localization, FAK was phosphorylated in periovulatory follicles. The injection of FAK inhibitor suppressed not only ovulation but also luteinization of granulosa cells and cumulus expansion. In cultured-granulosa cells, fibronectin-integrin synergistically activated FAK with amphiregulin (AREG). Such cooperative stimulations induced a morphological change in granulosa cells, which resulted in the maximum level of progesterone production via the induction of <i>Hsd3b</i>. When cumulus-oocyte complexes (COCs) were cultured with AREG in the presence of serum, the maximum level of cumulus expansion was observed. The AREG-induced cumulus expansion was also suppressed by FAK inhibitor. Thus, it is concluded that fibronectin and AREG synergistically activate FAK not only in granulosa cells and cumulus cells to induce successful ovulation process.</p></div

Effects of FAK inhibitor Y15 during ovulation <i>in vivo</i>.

<p>(A): Ovarian morphology of mice treated with hCG for 8 or 16 h was observed by hematoxylin-eosin staining (n = 3 ovaries in each group at each time point). Y15: eCG-primed mice co-injected with hCG and Y15 (30 mg/kg). Scale bar is 100 μm. (B): Effect of Y15 on cumulus expansion (surface area of COCs in periovulatory follicles of the ovary). Ovaries were collected at 0, 8, and 10 h after hCG injection with or without Y15 (30 mg/kg). The ovarian tissue was stained with hematoxylin-eosin, and individual areas of COCs were measured in periovulatory follicles in sections containing an oocyte with a nucleus. C (control): eCG-primed mice injected with hCG; Y15: eCG-primed mice co-injected with hCG and Y15 (30 mg/kg). *; Treatment with Y15 significantly decreased the mean surface area of COCs compared with the control at each time point (p<0.05). Values are the mean ± SEM of five COCs each in three sections. (C): The number of ovulated oocytes when mice were co-administered with hCG and Y15. Ovulated oocytes were collected from oviducts and counted at 16 h after hCG injection. Values are the mean ± SEM of five mice. *; Significant differences were observed compared with the control (p<0.05). C (control): Immature female mice treated with eCG followed by hCG stimulation; Y15: mice co-injected with hCG and Y15 (30 mg/kg) at 48 h after eCG injection. (D): Expression of genes involved in progesterone production and hyaluronic acid synthesis in the ovary of mice treated with hCG and Y15 (30 mg/kg). Three mice were used for sampling granulosa cells in each treatment group at each time point. The mRNA levels of <i>Star</i>, <i>Cyp11a1</i>, <i>Hsd3b1</i>, <i>Ptgs2</i> and <i>Has2</i> were analyzed by real-time PCR and normalized to that of <i>L19</i>. Values are the mean ± SEM of three replicates. *; Y15 treatment significantly suppressed the expression of each gene compared with the control at each time point (p<0.05).</p

Expression of fibronectin and integrins in the mouse ovary during ovulation.

<p>Localization of fibronectin and integrin α5 in the mouse ovary was detected by immunofluorescence staining (n = 3 mice in each time point). Proteins were visualized with Cy3 (fibronectin) and FITC (integrin α5). The nucleus was counterstained with DAPI. Scale bar is 100 μm.</p

Synergistic effects of fibronectin/serum coating and AREG stimulation on the morphology of granulosa cells <i>in vitro</i>.

<p>(A): Granulosa cells were collected from the ovary of mice treated with eCG for 48 h and cultured in uncoated wells (upper panel, Free) or serum-coated wells (lower panel, serum) with (right panel, AREG) or without (left panel, control) AREG. After 24 h, the cultured granulosa cells were stained for F-actin and counterstained with DAPI. Scale bar is 10 μm. (B): Surface area of granulosa cells cultured for 24 h with or without AREG on uncoated or serum-coated wells. The surface area of each granulosa cell was measured by an area measurement system associated with the BZ-9000 microscope. Values are the mean ± SEM of fifteen granulosa cells in each treatment group of three independent experiments (45 granulosa cells in total were measured in each treatment group). Data were statistically analyzed by two-way ANOVA. Because the significant difference was observed for each factor, the comparison analysis was performed by the Student’s t-test. *; AREG significantly increased the area of granulosa cells cultured on serum-coated wells (p<0.05). Free: granulosa cells cultured on uncoated wells; serum: granulosa cells cultured on serum-coated wells; C (control): granulosa cells treated without AREG; AREG: granulosa cells treated with AREG. (C): The number of actin stress fiber in each granulosa cell cultured on serum-coated well or free well with or without AREG. Values are the mean ± SEM of fifteen granulosa cells. *; Significant differences were observed compared with the control (p<0.05).</p

Roles of AREG and fibronectin in progesterone production of granulosa cells.

<p>(A): Progesterone concentrations in culture supernatants of granulosa cells treated for 24 h with or without AREG on uncoated or fibronectin-coated wells. Values are the mean ± SEM of three replicates. Data were statistically analyzed by two-way ANOVA. Because the significant difference was observed for each factor, the comparison analysis was performed by the Student’s t-test. *; AREG significantly increased the area of granulosa cells cultured on fibronectin-coated wells or free wells (p<0.05). Free: granulosa cells cultured on uncoated wells; Fibronectin: granulosa cells cultured on fibronectin-coated wells; C (control): granulosa cells treated without AREG; AREG: granulosa cells treated with AREG. (B): Expression of genes involved in progesterone production (<i>Star</i>, <i>Cyp11a1</i>, and <i>Hsd3b1</i>) in granulosa cells treated for 24 h with or without AREG on uncoated or fibronectin-coated wells. Values are the mean ± SEM of three replicates. Data were statistically analyzed by two-way ANOVA. Because the significant difference was observed for each factor, the comparison analysis was performed by the Student’s t-test. *; AREG significantly increased the area of granulosa cells cultured on fibronectin-coated wells or free wells (p<0.05). **AREG significantly increased the area of granulosa cells cultured on fibronectin-coated wells (p<0.05). Free: granulosa cells cultured on uncoated wells; Fibronectin: granulosa cells cultured on fibronectin-coated wells; C (control): granulosa cells treated without AREG; AREG: granulosa cells treated with AREG. (C): Additional effects of Y15 on AREG-induced progesterone production in granulosa cells cultured on fibronectin-coated wells or free-wells. Values are the mean ± SEM of three replicates. *; Y15 treatment significantly decreased the progesterone level when granulosa cells were cultured on fibronectin-coated wells (p<0.05). (D): The treatment with Y15 inhibited AREG-induced expression of genes involved in progesterone production (<i>Star</i>, <i>Cyp11a1</i>, and <i>Hsd3b1</i>) in granulosa cells. Values are the mean ± SEM of three replicates. *; Y15 treatment significantly inhibited the gene expression levels compared with each control (p<0.05).</p

Effects of FAK inhibitor Y15 and RGD peptide on cumulus expansion <i>in vitro</i>.

<p>(A,B): The surface area of expanded COCs was measured using the BZ-9000 microscope. COCs were isolated from ovaries of mice primed with eCG for 48 h. Non-expanded COCs were selected, and 50 COCs were cultured in a 50 μl drop of defined medium containing 1% FBS and 100 ng/ml AREG. Some COCs were treated with AREG and 100 nM Y15 or 100 μM RGD peptide (RGD; arginine-glycine-aspartate, tripeptide). After 16 h, the area of each expanded COC was measured using the BZ-9000 microscope. Scale bar is 100 μm. The left panel shows an image of an expanded COC in each treatment group after culture. Values are the mean ± SEM of fifteen COCs each in three replicates. *; Y15 or RGD peptide significantly suppressed cumulus expansion compared with the control (p<0.05). (C): The rate of metaphase II stage oocytes when COCs were cultured with Y15 or RGD peptide. *; Y15 or RGD peptide significantly decreased the rate of metaphase II (M2) oocytes compared with the control (p<0.05). (D): Expression of genes involved in cumulus expansion (<i>Has2</i>, <i>Tnfaip6</i>, and <i>Ptx3</i>). Each value is presented as the gene expression level. Values are the mean ± SEM of three replicates.</p

Expression of phosphorylation of FAK in the mouse ovary during ovulation.

<p>(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. The ovary was collected from mice treated with hCG. Three mice were used for ovarian sampling at each time point. β-actin was used as a loading control. The intensity of the bands was analyzed and values are mean +/- SEM of 3 replicates. *; Significant differences were observed by hCG injection as compared with those before hCG injection (0h) (P<0.05). (B): Phosphorylation status of FAK in granulosa cells cultured for 4 h with or without AREG on uncoated or fibronectin-coated plates. C (control): granulosa cells cultured without AREG; AREG: granulosa cells treated with 100 ng/ml AREG; AG: granulosa cells treated with both AREG and 10 μM AG1478 (AG, a selective EGFR tyrosine kinase inhibitor); Free: granulosa cells cultured on uncoated wells; Fibronectin: granulosa cells cultured on fibronectin-coated wells. β-actin was used as a loading control. The intensity of the bands was analyzed and values are mean +/- SEM of 3 replicates. *; Significant differences were observed by the addition of AG1478 (AG) (P<0.05). **; The treatment with AREG significantly increased the intensity of pFAK (Tyr925) in granulosa cells cultured on the fibronectin-coated wells (P<0.05).</p