HAS2-AS1 is a novel LH/hCG target gene regulating HAS2 expression and enhancing cumulus cells migration.

BACKGROUND: The cumulus expansion process is one of the LH mediated ovulatory processes. Hyaluronan synthase 2 (HAS2) regulates the synthesis of hyaluronic acid, the main component of the cumulus expansion process. Recently, the lncRNA HAS2 antisense RNA 1 (HAS2-AS1) was identified in our global transcriptome RNA-sequencing of novel ovulation associated genes. The role of HAS2-AS1 in HAS2 regulation w.as studied previously with contradictive results in different models but not in the ovary. Taken together the induction of HAS2-AS1 and the important role of HAS2 in the cumulus expansion process, we hypothesize that HAS2-AS1 regulate HAS2 expression and function in the ovary. Therefore we undertook to study the expression, regulation, and possible functional role of HAS2-AS1 in the human ovary. RESULTS: HAS2-AS1, located within the HAS2 gene that was highly regulated in our library. We found that HAS2-AS1 express mainly in cumulus cells (CCs). Furthermore, HAS2-AS1 showed low expression in immature CCs and a significant increase expression in mature CCs. Functional studies reveal that inhibition of HAS2-AS1 by siRNA caused decrease expression of HAS2. Furthermore, inhibition of HAS2-AS1 by siRNA results in decrease migration of granulosa cells. CONCLUSIONS: Our results suggest that HAS2-AS1 is an LH/hCG target gene that plays a positive role in HAS2 expression and thus might play a role in regulating cumulus expansion and migration.

An optimized model for hCG stimulation of human mural granulosa cell culture.

Ovarian follicular development and ovulation in mammals is a highly-regulated process. Most of the current knowledge of ovarian processes was obtained from the studies of non-human models. Molecular studies on human ovarian processes suffer from lack of material and appropriate research tools. Mural granulosa cells (MGCs) culture is a major tool for studying the effect of different substances but a major problem for using these primary MGCs is their unresponsiveness to hCG stimulation at the time of oocyte retrieval. It is acceptable that MGCs regain responsiveness during days in culture but when the best time is and how to accelerate the regenerative process are unknown. The aim of the current study was to establish an optimized protocol which will provide a practical and efficient tool to examine the effect of LH/hCG on different downstream targets in luteinized MGCs. hCG effects were examined according to days in culture and hCG stimulation time. As read-out, we analyzed the gene expression of known hCG targets, protein production, and progesterone secretion. Our results show that with a daily medium exchange, the strongest effect was achieved already 4 days after seeding. On day 4, hCG stimulation triggers two major patterns of gene expression. Early induced genes were highly expressed 6-8 h after hCG, while 24 h of hCG stimulation was needed for late induced genes. Based on our results, we suggest daily medium exchange for 4 days before adding hCG and examine its effect 6 and 24 h later.

Differential regulation of cumulus cell transcription during oocyte maturation in vivo and in vitro.

Differences in cumulus cell gene expression after oocyte maturation in vitro (IVM) or in vivo have been described in previous studies. However, the possible impact of follicle stage on gene expression deregulation during human oocyte IVM remains unknown. Expression of selected genes of interest was compared in cumulus cell of three classes of human cumulus cell-oocyte complexes (COCs): a) COCs derived from human chorionic gonadotropin (hCG)-triggered IVM cycles, collected at the germinal vesicle (GV) stage from mid-sized follicles (4-12 mm) and matured in vitro (IVM-GV); b) COCs derived from hCG-triggered IVM cycles, collected from mid-sized follicles (4-12 mm) and matured in vivo (IVM-MII); c) COCs derived from controlled ovarian stimulation in vitro fertilization (IVF) cycles, collected from large/preovulatory follicles and matured in vivo (IVF-MII). Overall, mRNA levels of the large majority of the 20 genes of different regulative and metabolic pathways subject to analysis were altered in IVM samples compared with in vivo matured COCs. In some cases, follicle size appeared to have a role in determining transcription deregulation. For example, in comparison to the IVF-MII control, the luteinizing hormone receptor was largely overexpressed in both IVM-GV and IVM-MII COCs, therefore irrespective of IVM. However, in other circumstances follicle size and IVM had distinct and opposite impacts on gene expression, as shown by transcription of amphiregulin, which was increased in IVM-MII COCs, but decreased in COCs matured in vitro (IVM-GV) compared with the IVF-MII control. This study confirms and extends previous data on gene expression dysregulation during IVM and indicates that the size of follicles from which immature oocytes are retrieved can be an independent factor of differential transcriptional regulation.

Dibutyl phthalate impairs steroidogenesis and a subset of LH-dependent genes in cultured human mural granulosa cell in vitro.

Exposure to di-butyl phthalate (DBP) exerts negative effects on female fertility in animal models, but human studies remain limited. Here, the effects of DBP exposure on mural granulosa cell function were investigated in primary cultures from women undergoing in vitro fertilization. Cultured cells treated with various doses of DBP (0, 0.01µg/mL, 0.1µg/mL, 1µg/mL, 10µg/mL, or 100µg/mL) for 48h were assessed using enzyme-linked immunosorbent assay and qRT-PCR. Treatment with 100µg/mL DBP resulted in significantly lower 17ß-estradiol and progesterone production (p<0.01). It also resulted in altered mRNA expression of steroidogenic, angiogenic, and epidermal growth factor-like growth factor genes: CYP11A1 (p<0.001), CYP19A1 (aromatase) (p<0.001), VEGF-A (p<0.02), BTC (p=0.009), and EREG (p=0.04). StAR expression was impaired after exposure to both 10 and 100µg/mL (p<0.03 and p<0.001, respectively). Our results indicate that in vitro exposure of granulosa cells to high doses of DBP alters cell functions.

Standard human chorionic gonadotropin versus double trigger for final oocyte maturation results in different granulosa cells gene expressions: a pilot study.

OBJECTIVE: To investigate the messenger RNA (mRNA) expression of reproduction-related genes in granulosa cells (GCs) of patients triggered with hCG compared with patients triggered with GnRH agonist and hCG (double trigger) for final oocyte maturation. DESIGN: Granulosa cells were obtained at the time of oocyte retrieval, and gene expression was analyzed using quantitative real-time polymerase chain reaction. SETTING: Referral center. PATIENT(S): Fifteen women undergoing controlled ovarian hyperstimulation for IVF who received hCG for final follicular maturation and in a subsequent IVF cycle received double trigger. INTERVENTION(S): Granulosa cells collection. MAIN OUTCOME MEASURE(S): The expression of genes related to ovarian hyperstimulation syndrome, gap junction, and epidermal-like growth factor in GCs. RESULT(S): The mRNA expressions of amphiregulin (2.1 vs. 1, arbitrary unit) and epiregulin (2.5 vs. 1, arbitrary unit) were significantly higher in the double trigger group compared with the hCG group. We found no difference in luteinizing hormone receptor and follicle stimulating hormone receptor mRNA expressions between the two groups. Moreover, although the mRNA expression of pigment epithelium-derived factor (1.5 vs. 1, arbitrary unit) was significantly higher in the double trigger group, no between-group differences were observed in the expression of vascular endothelial growth factor and GnRH receptor. The mRNA expression of conexin43 in cumulus cells (0.7 vs. 1, arbitrary unit) was significantly lower in the double trigger group compared with the hCG group. CONCLUSION(S): Our findings suggest that the decreased expression of conexin43 and the increased expression of epiregulin and amphiregulin in the GCs from patients receiving the double trigger may explain the suggested improved oocyte and embryo quality related to the double triggering group.

Differential expression of poliovirus receptor, regulator of G-protein signaling 11 and erythrocyte protein band 4.1-like 3 in human granulosa cells during follicular growth and maturation.

Poliovirus receptor (PVR), regulator of G-protein signaling-11 (RGS11), and erythrocyte protein band-4.1-like 3 (EPB41L3) have been proposed to function in follicular maturation in mouse models. We have examined their expression in human mural (mGCs) and cumulus granulosa cells (CCs). Expression of PVR and RGS11 in mGCs decreased in medium-sized follicles compared to small follicles of IVM cycles and increased again in large follicles. Luteinization caused decreased expression of both PVR and RGS11. In vitro incubation of mGCs with progesterone-rich conditioned media decreased expression of RGS11 without affecting PVR levels. Inhibition of progesterone signaling enhanced expression of both RGS11 and PVR. Expression in CCs was examined by means of global transcriptome sequencing analysis RGS11 and EPB41L3 increased in CCs during follicular maturation while PVR levels did not change. In conclusion, during human follicular maturation there are significant changes in expression of PVR, RGS11 and EPB41L3, possibly regulated by progesterone.

GnRH agonist vs. hCG for triggering of ovulation--differential effects on gene expression in human granulosa cells.

OBJECTIVE: To investigate the mRNA expression of genes related to steroidogenesis and OHSS in granulosa cells (GCs) of patients triggered with GnRH agonist compared to patients triggered with hCG. DESIGN: Mural GCs were obtained at the time of oocyte retrieval and gene expression was analyzed using quantitative real time RT-PCR. SETTINGS: Single center, case control study. PATIENT(S): 24 women who were treated with GnRH agonist or hCG for triggering of ovulation. INTERVENTIONS: GC collection. MAIN OUTCOME MEASURE(S): The expression of genes related to steroidogenesis and OHSS in mural GCs. RESULTS: The fertilization rate was similar in the two groups. The mRNA expression of CYP19A1 (0.50 vs 1, arbitrary unit), CYP11A1 (0.6 vs. 1) and 3 beta hydroxysteroid-dehydrogenase (0.39 vs 1) was significantly lower in the GnRH group. The expression of VEGF (0.74 vs. 1) and inhibin ß B (0.38 vs 1) was lower in the GnRH analog triggered group. CONCLUSION: Expression of genes related to steroidogenesis is lower at the time of oocyte retrieval in patients triggered with GnRH agonist. The decreased expression of VEGF and inhibin ß B in the GnRH agonist group can explain the mechanism of early OHSS prevention.

Progesterone antagonist, RU486, represses LHCGR expression and LH/hCG signaling in cultured luteinized human mural granulosa cells.

Progesterone, the main steroid synthesized by the corpus luteum (CL), prepares the uterus for implantation, maintains the CL survival, and induces progesterone auto-secretion. However, the molecular mechanisms involving the progesterone auto-secretion pathways at the luteal phase are not fully understood, especially in humans. We aim to study the molecular mechanism of the progesterone pathway in human granulosa cells. Our model system consists of luteinized human-mural-granulosa-cells (hmGCs) obtained from follicles aspirated during in vitro fertilization (IVF) procedures. hmGCs were seeded in culture and were subjected to different hormonal treatments. mRNA levels were analyzed by quantitative real-time PCR (qRT-PCR). Progesterone levels were measured by enzyme immunoassay (EIA). We show that exposure of luteinized hmGCs to the progesterone receptor antagonist, RU486 (mifepristone), resulted in inhibition of LHCGR, LH/hCG target genes and progesterone secretion. Exposure of hmGCs to medium that was incubated with hmGCs for 4 d - conditioned medium (CM), which contain 150 ± 7.5 nM progesterone, resulted in induction of LHCGR and LH/hCG target genes, which was blocked by RU486. In addition, RU486 inhibited some of the progesterone biosynthesis pathway genes. Our results revealed a novel mechanism of the progesterone antagonist pathway in the luteal granulosa cells and emphasis the fundamental role of progesterone in the early luteal phase.