Oocyte-expressed interleukin 7 suppresses granulosa cell apoptosis and promotes oocyte maturation in rats.

Development of ovarian follicles is regulated by pituitary-derived gonadotropins together with local ovarian paracrine factors. Based on DNA microarray data, we performed RT-PCR and immunostaining to demonstrate the expression of interleukin 7 transcripts in oocytes of preantral, antral, and preovulatory follicles in rats. We also found the expression of interleukin 7 receptor and the coreceptor interleukin 2 receptor gamma in granulosa cells, cumulus cells, and preovulatory oocytes. In cultured rat granulosa cells obtained from early antral and preovulatory follicles, treatment with interleukin 7 stimulated the phosphorylation of AKT, glycogen synthase kinase (GSK3B), and STAT5 proteins in a time- and dose-dependent manner. Furthermore, measurement of mitochondrial reductase activity indicated that treatment with interleukin 7, similar to gonadotropins, increased the number of viable granulosa cells during a 24-h culture period. Furthermore, monitoring of the activities of apoptotic enzymes (caspase 3/7) indicated that treatment with interleukin 7 suppressed apoptosis of cultured granulosa cells from both antral and preovulatory follicles following serum withdrawal. The apoptosis-suppressing actions of interleukin 7 were blocked by an inhibitor of the phosphoinositol-3-kinase (PIK3)/AKT pathway. Furthermore, treatment of cultured preovulatory follicles with interleukin 7, like treatment with human chorionic gonadotropin, induced germinal vesicle breakdown of oocytes. The stimulatory effect of interleukin 7 was also blocked by inhibitors of the PIK3/AKT pathway. The present findings suggest that oocyte-derived interleukin 7 could act on neighboring granulosa cells as a survival factor and promote the nuclear maturation of preovulatory oocytes through activation of the PIK3/AKT pathway.

Suppression of progesterone production by stresscopin/urocortin 3 in cultured human granulosa-lutein cells.

BACKGROUND: Corticotropin-releasing hormone (CRH) and its receptors have been identified in female reproductive tissues. CRH regulates follicular maturation, ovulation, luteolysis and steroidgenesis. A CRH-related peptide stresscopin (SCP), or urocortin III (Ucn3), has recently been identified, but its functions in the ovary remain to be elucidated. In the present study, we investigated the effects of SCP/Ucn3 on progesterone production in cultured human granulosa-lutein cells. METHODS: The presence of SCP/Ucn3 and CRH type-2 receptor (CRHR2) in cultured granulosa-lutein cells from 21 infertile women (aged 22-36 years) was examined by RT-PCR and immunocytochemistry. The concentration of SCP/Ucn3 in follicular fluid, human serum and culture medium was examined by radioimmunoassay. Progesterone production by cultured granulosa-lutein cells treated with SCP/Ucn3 was examined by enzyme-linked immunosorbent assay. RESULTS: SCP/Ucn3 and CRHR2 mRNAs and proteins were expressed in granulosa-lutein cells. SCP/Ucn3 was detected in culture media of granulosa-lutein cells and follicular fluid. Treatment of cultured granulosa-lutein cells with 0.1, 1.0 or 10 nM SCP/Ucn3 decreased progesterone secretion when compared with untreated control (all P < 0.05). Concomitant treatment with the CRHR2 antagonist antisauvagine-30 counteracted the inhibitory effects of SCP/Ucn3 on progesterone secretion, suggesting a mediatory role of CRHR2. CONCLUSIONS: The present results suggest that the SCP/CRHR2 system is present in human ovaries and treatment with SCP/Ucn3 inhibits progesterone production by cultured granulosa-lutein cells through interaction with CRHR2.

Effects of corticotropin-releasing hormone and stresscopin on vascular endothelial growth factor mRNA expression in cultured early human extravillous trophoblasts.

Corticotropin-releasing hormone (CRH) takes a role in the regulation of the onset of parturition. Stresscopin (SCP) is a high affinity ligand for CRH receptor (CRHR)-2. CRHR-2 inhibits VEGF-induced neovascularization. In the present study, we investigated the effects of CRH and SCP on VEGF expression in early placental extravillous trophoblasts (EVTs). Isolation and culture of trophoblasts differentiating into EVTs were performed by the enzymatic digestion of anchoring early placental villi. The presence of CRH, SCP, CRHR-1, and CRHR-2 in cultured EVTs was examined by RT-PCR and immunocytochemistry. The effects of CRH and SCP on VEGF mRNA levels in cultured EVTs were assessed by real-time RT-PCR. CRH, SCP, CRHR-1, and CRHR-2 were expressed in cultured EVTs at mRNA and protein levels. Treatment with either 100 nM CRH or 100 nM SCP for 24 h decreased VEGF mRNA levels in cultured EVTs. The CRH- and SCP-induced decrease in VEGF mRNA levels was counteracted by the concomitant treatment with CRHR-2 antagonist antisauvagine-30, but not with CRHR-1 antagonist antalarmin. We demonstrated that CRH and SCP inhibited VEGF mRNA expression in cultured EVTs through the interaction with CRHR-2, suggesting that CRH and SCP may inhibit angiogenesis during early placentation.

Effects of recombinant H2 relaxin on the expression of matrix metalloproteinases and tissue inhibitor metalloproteinase in cultured early placental extravillous trophoblasts.

Relaxin promotes softening of the uterine cervix and inhibits uterine contractility in rats, mice and pigs. Little information, however, is available about the role of relaxin in humans. In 2002, LGR7 and LGR8 were discovered to be receptors for relaxin and those receptors were identified in the human placenta. Thus, in this study, effects of recombinant H2 (rH2) relaxin on human early placental extravillous trophoblasts (EVTs) were examined. Isolation of EVTs from early placental trophoblasts was performed using the procedures established in our laboratory. After 48-h subculture, the presence of relaxin receptors in cultured EVTs was characterized by RT-PCR and immunoblotting. The cultured EVTs were treated with different doses (0.3-3 ng/ml) of rH2 relaxin for 24 h. The effects of rH2 relaxin on MMP-2, -3, -9 and TIMP-1 mRNAs levels were examined by real-time RT-PCR. RT-PCR and immunoblotting revealed that relaxin receptors are present in early placental EVTs. Treatment with rH2 relaxin increased MMP-2 and -9 mRNAs levels and decreased TIMP-1 mRNA levels in cultured EVTs, whereas rH2 relaxin did not affect MMP-3 mRNA levels. These results suggest that relaxin may promote the invasive potential of early placental EVTs through up-regulating MMP-2, -9 mRNAs and down-regulating TIMP-1 mRNA in EVTs.