Mural Granulosa Cells Research Articles

Cooperative effects of oocytes and estrogen on the forkhead box L2 expression in mural granulosa cells in mice

Forkhead box L2 (FOXL2) plays a critical role in the development and function of mammalian ovaries. In fact, the causative effects of FOXL2 misregulations have been identified in many ovarian diseases, such as primary ovarian insufficiency and granulosa cell tumor; however, the mechanism by which FOXL2 expression is regulated is not well studied. Here, we showed that FOXL2 expression in ovarian mural granulosa cells (MGCs) requires stimulation by both oocyte-derived signals and estrogen in mice. In the absence of oocytes or estrogen, expression of FOXL2 and its transcriptional targets, Cyp19a1 and Fst mRNA, in MGCs were significantly decreased. Moreover, expression levels of Sox9 mRNA, but not SOX9 protein, were significantly increased in the FOXL2-reduced MGCs. FOXL2 expression in MGCs was maintained with either oocytes or recombinant proteins of oocyte-derived paracrine factors, BMP15 and GDF9, together with estrogen, and this oocyte effect was abrogated with an ALK5 inhibitor, SB431542. In addition, the FOXL2 level was significantly decreased in MGCs isolated from Bmp15−/− /Gdf9+/− mice. Therefore, oocyte, probably with estrogen, plays a critical role in the regulation of FOXL2 expression in mural granulosa cells in mice.

Intrafollicular injection of nanomolecules for advancing knowledge on folliculogenesis in livestock

Despite the progress in assisted reproductive techniques, there is still a lack of rapid and minimally invasive in situ approaches for further enhancements of female fertility. Therefore, we synthesized clinically relevant liposome nanoparticles for ovarian intrafollicular injection to allow in vivo cellular imaging for future drug delivery, using the mare as an animal model. Ovarian follicles of living mares were injected in vivo with fluorescently labeled liposomes. Samples of the follicular wall (mural granulosa, theca interna, and theca externa), granulosa cells, and follicular fluid were harvested 24 h post-injection through the follicle wall biopsy (FWB), flushing, and aspiration techniques, respectively, using a transvaginal ultrasound-guided approach. In parallel, post-mortem dissected, and cultured porcine antral follicles were microinjected with doxorubicin-encapsulated liposomes to assess intracellular delivery potential. All injected mare and pig follicles were macroscopically healthy, and fluorescence imaging revealed successful intrafollicular binding to mural granulosa cells and progressive migration of liposomes to other follicle cell layers (theca interna, and theca externa), regardless of the follicle size. Intracellular delivery of doxorubicin was confirmed in all porcine follicle wall cell types. We conclude that the intrafollicular injection of nanomolecules is a promising approach for real-time monitoring of intrafollicular processes and potential utilization of in vivo cellular drug delivery to assist in follicle disease treatments and fertility improvement.

Dysregulated sphingolipid metabolism and autophagy in granulosa cells of women with endometriosis.

We evaluated metabolic profiles between cumulus cells (CCs) and mural granulosa cells (MGCs) derived from women with endometriosis to identify their correlations with oocyte quality. CCs and MGCs were collected from women with and without endometriosis undergoing in vitro fertilization/intracytoplasmic sperm injection treatment. The metabolomics of CCs and MGCs were measured by liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) followed by a quantitative polymerase chain reaction to further confirm the genes involved in the metabolic results. LC-MS/MS analysis revealed differences in 24 metabolites of CCs and 71 metabolites of MGCs between groups. Among them, five metabolites were upregulated and 19 metabolites were downregulated in CCs with endometriosis, whereas three metabolites were upregulated and 68 metabolites were downregulated in MGCs with endometriosis. Metabolites related to sphingolipid metabolism, which included palmitic acid (PA) and docosahexaenoic acid, increased significantly only in CCs with endometriosis, whereas sphingosine and PA were significantly downregulated in MGCs with endometriosis compared with CCs and MGCs without endometriosis. Gene expression involved in ceramide synthesis (CERS1, SPTL1, and SMPD1) and autophagy (BECN1, LAMP, and PC3) were significantly higher in CCs with endometriosis according to FASN, BECN1, and LAMP protein expressions. However, gene expression involved in ceramide synthesis (SPHK1, ASAH1, and SGPP1) and autophagy (BECN1, LAMP, and PC3) were significantly lower in MGCs with endometriosis, whereas CERS1 and UGCG expression increased. There are differences in sphingolipid metabolites in CCs and MGCs with endometriosis compared with women without endometriosis. These differences seem to be involved in the regulation of autophagic cell death in preovulatory follicles.

Heat-induced increases in body temperature in lactating dairy cows: impact on the cumulus and granulosa cell transcriptome of the periovulatory follicle.

Cows acutely heat stressed after a pharmacologically induced luteinizing hormone (LH) surge had periovulatory changes in the follicular fluid proteome that may potentiate ovulation and impact oocyte developmental competence. Because the cellular origins of differentially abundant proteins were not known, we have examined the cumulus and granulosa cell transcriptomes from the periovulatory follicle in cows exhibiting varying levels of hyperthermia when occurring after the LH surge. After pharmacological induction of a dominant follicle, lactating dairy cows were administered gonadotropin releasing hormone (GnRH) and maintained in thermoneutral conditions (~67 temperature-humidity index [THI]) or heat stress conditions where THI was steadily increased for ~12 h (71 to 86 THI) and was sufficient to steadily elevate rectal temperatures. Cumulus-oocyte complexes and mural granulosa cells were recovered by transvaginal aspiration of dominant follicle content ~16 h after GnRH. Rectal temperature was used as a continuous, independent variable to identify differentially expressed genes (DEGs) increased or decreased per each 1 °C change in temperature. Cumulus (n = 9 samples) and granulosa (n = 8 samples) cells differentially expressed (false discovery rate [FDR] < 0.05) 25 and 87 genes, respectively. The majority of DEGs were upregulated by hyperthermia. Steady increases in THI are more like the "turning of a dial" than the "flipping of a switch." The moderate but impactful increases in rectal temperature induced modest fold changes in gene expression (<2-fold per 1 °C change in rectal temperature). Identification of cumulus DEGs involved in cell junctions, plasma membrane rafts, and cell-cycle regulation are consistent with marked changes in the interconnectedness and function of cumulus after the LH surge. Depending on the extent to which impacts may be occurring at the junctional level, cumulus changes may have indirect but impactful consequences on the oocyte as it undergoes meiotic maturation. Two granulosa cell DEGs have been reported by others to promote ovulation. Based on what is known, several other DEGs are suggestive of impacts on collagen formation or angiogenesis. Collectively these and other findings provide important insight regarding the extent to which the transcriptomes of the components of the periovulatory follicle (cumulus and mural granulosa cells) are affected by varying degrees of hyperthermia.

P-516 Cell-type specific role of Fragile X Mental Retardation polyglycine protein in blood of women with Fragile X Mental Retardation 1 gene premutation

Abstract Study question What cell type is specific for the expression of FMRpolyG of FMR1 premutation (PM) in peripheral mononuclear blood cells (PMBC) and how can this impact female fertility? Summary answer The expression of FMRpolyG is significantly higher in T cells from peripheral blood mononuclear cells (PBMCs) of FMR1 PM carriers. What is known already Fragile X-associated primary ovarian insufficiency (FXPOI) is characterized by oligo/amenorrhea and hypergondotropic hypogonadism and is caused by expansion of the CGG-repeat in the 5'UTR of FMR1. Autoimmunity is highly associated with POI and in FMR1-PM activated inflammatory state and immune response have been discovered. RAN-translation dependent on variable CGG-repeat length is thought to cause FXPOI due to production of the polyglycine-containing-FMR1-protein, FMRpolyG. Colocalization of ubiquitin and FMRpolyG was recently demonstrated in ovarian stromal and mural granulosa cells from FMR1 PM carriers. Our previous data have also shown that FMRpolyG is aggregated in ubiquitin-positive inclusions in PBMCs from PM carriers. Study design, size, duration PBMCs and granulosa cells (GCs) from women with PM (5) and women without PM (10) (controls) were examined by immunofluorescence staining (IF) for the presence of inclusions positive for ubiquitin and FMRpolyG. Cell lysis and protein extraction samples were subjected to Western blot analysis (WB) to detect FMRP and FMRpolyG. Flow cytometric analyzes (FACs) were performed to determine cell type-specific expression of FMRpolyG. Participants/materials, setting, methods PBMCs were isolated from peripheral blood using Ficoll-Paque. PBMCs were fixed, IF stained for FMRpolyG and ubiquitin, and analyzed by fluorescence microscopy. WB was used to detect the expression of FMRP, FMRpolyG in extracted protein from lymphocytes and GCs. PBMCs were surface stained with CD3, CD14 and CD19 antibodies and later intracellular stained with FMRpolyG and ubiquitin for FACs analysis. Main results and the role of chance FMRpolyG aggregates were detected as ubiquitin-positive inclusions in PBMCs from PM carriers, whereas only weak signal without inclusions was detected in controls. We detected FMRpolyG as a 15- to 25-kDa protein in PBMCs from two FMR1 PM carriers with 124 and 81 CGG repeats, a PM range that is supposed to carry the highest risk to develop FXPOI within all PM carriers. Using FACs, we found that the expression levels of FMRpolyG were significantly higher in the FMR1 PM carriers than in the group without PM (3.5-fold, p = 0.03). Both FMR1 PM and non-PM groups showed normal distribution of T cells (58.38% ± 11.54 and 58.72% ± 6.16, respectively). However, FMRpolyG fluorescence intensity was 28.98-fold higher (p = 0.01) in the T cells of FMR1 PM carriers than in those of women without PM. FMRpolyG expression levels of B cells (CD19+) and monocytes (CD14+) were comparable in both groups. Of note, the ubiquitin-positive cells of FMR1 PM carriers had significantly higher expression levels of FMRpolyG than those of non-PM carriers (17.84-fold, p &amp;lt; 0.0001). Limitations, reasons for caution More patients are needed to support the present results. Further studies on the possible consequences of these FMRpolyG-positive inclusions in PM carriers are also advisable. Wider implications of the findings We found for the first time FMRpolyG aggregates in the peripheral blood of PM-carriers and a significant increase in FMRpolyG expression in T cells from PM-carriers. These results suggest that FMRpolyG may have a toxic potential and play an immunological role in ovarian damage, finally triggering the development of FXPOI. Trial registration number RE 3647/1-1 and /1-2

P-567 Recombinant hCG (choriogonadotropin beta, CG beta) exerts a positive dose-dependent effect on human follicular steroidogenesis during ovarian stimulation using a constant rFSH administration

Abstract Study question How does increasing doses of CG beta augment follicular steroidogenesis and is it associated with gene expression in cumulus cells? Summary answer With the exception of progesterone, CG beta exerts dose-dependent effects on intrafollicular steroid hormone concentrations and CYP19a1 expression during constant rFSH administration. What is known already CG beta (FE 999302) is a newly developed rhCG, produced by a human cell line (PER.C6®). This variant contains the same amino acid sequence as the endogenous hCG but presents with a different glycosylation profile when compared to urinary hCG or rhCG derived from a CHO cell line. CG beta has also been shown to have a longer half-life and higher relative potency. It is well known that hCG like LH creates an intracellular response through the LHR expressed on theca cells and mature granulosa cells and hereby augment the production of androgens, progesterone, and oestrogen. Study design, size, duration This study is part of a randomised, double-blind, placebo-controlled trial to investigate the efficacy and safety of CG beta as an add-on treatment to rFSH in women undergoing ovarian stimulation during a long GnRH agonist protocol. The primary endpoint of this study was intrafollicular steroid levels in relation to CG beta administration. Secondary outcomes were gene expression of LHR, FSHR, CYP19a1, and androgen receptor (AR) and single nucleotide polymorphisms (SNPs) in LHR(312) and FSHR(307). Participants/materials, setting, methods 619 women with AMH levels 5-35 pmol/L were randomized to receive either placebo or 1, 2, 4, 8, or 12 µg CG beta from day one of stimulation combined with a constant individualized dose of rFSH. Follicular fluid (FF) (n = 558), granulosa (n = 498) and cumulus cells (n = 368) were collected at oocyte retrieval. Steroid FF hormones were measured using ELISAs, gene expression was analysed in cumulus cells by qRT-PCR, and SNP analysis was performed in granulosa cells. Main results and the role of chance This study found that CG beta has a pronounced positive dose-dependent effect on intrafollicular concentrations of 17-OH-progesterone, androstenedione, testosterone, and oestradiol during constant rFSH administration. As compared to the placebo group without CG beta administration the concentrations of steroids were significantly dose-dependently increased (p-value&amp;lt;0.0001) reaching up to 10 times higher values in the highest dose group without a clear plateauing. However, for progesterone, there was no statistically significant difference between the CG beta dose groups and placebo. The gene expression level of CYP19a1 in the cumulus cells collected at oocyte retrieval increased significantly (p-value=0.0325) for the highest dose of CG beta. However, the gene expression level of FSHR, LHR, and AR in cumulus cells were not affected by CG beta administration, as no dose-response trend with increasing dose of CG beta was observed. Additionally, this study revealed that there was no overall clear difference in the number of oocytes retrieved between the different CG beta dose groups based on either the FSHR(307) or LHR(312) genotypes. Limitations, reasons for caution The gene expression was assessed in cumulus cells, but it would also have been interesting to evaluate the mural granulosa cells. Analysis of the gene expression of 3βHSD and other important steroidogenic enzymes in the mural granulosa cells could reveal further explanation to the effect of CG beta administration. Wider implications of the findings To our knowledge, this is the first study to show a clear dose-dependent effect of rhCG on human steroidogenesis during constant rFSH administration in preovulatory follicles. This reflects the importance of the combined effect of FSH and hCG/LH on granulosa cell activity, follicle health, and potentially oocyte quality. Trial registration number 000289

P-591 Cellular heterogeneity in the preovulatory follicle correlates with response to stimulation with recombinant FSH and affects somatic cell gene expression levels

Abstract Study question What is the difference in the composition and gene expression of pre-ovulatory follicular somatic cells between normo- and hyporesponder patients to rFSH stimulation? Summary answer Gene expression changes in the somatic cells of the preovulatory follicles of hyporesponder patients is partly attributable to distinct cell populations. What is known already Some women undergoing IVF respond suboptimally to the preceding ovarian stimulation without an indication of poor ovarian insufficiency nor advanced age. The gene expression levels in the somatic cells of their pre-ovulatory follicles have not been determined. However, the somatic cells play key roles in the transfer of gonadotrophin signals and in steroidogenesis. In the preovulatory follicle, being &amp;gt;20 mm in size, the somatic cells are exposed to various molecular gradients that drive their differentiation. Due to advancements in sequencing technologies the importance of somatic cell sub-populations in the etiologies of ovarian infertility can now be investigated by bioinformatic methods. Study design, size, duration Consecutive IVF patients &amp;lt;41 years of age were recruited at Nova Vita Clinic, Estonia, undergoing the antagonist stimulation protocol with rFSH administration. Ovarian puncture was performed for all patients after 36 hours of hCG stimulaton. rFSH dose &amp;gt;200 IU per one retrieved oocyte was considered as an indication for hyporesponse. Ten normo- and 9 hyporesponder patients were enrolled. Participants/materials, setting, methods Cells from the follicular fluid devoid of the cumulus oocyte complex were collected by centrifugation. RNA-seq was performed on the pooled cells of all participants. The study groups were compared by differential gene expression analysis by DESeq2. Single-cell RNA-seq was performed for &amp;gt;6000 individual cells of 3 normoresponder patients and cellular sub-populations were determined with Seurat package. CIBERTSORTx software was used to deconvolute the proportion of cell types from bulk RNA-seq data of all participants. Main results and the role of chance Alterations in the gene expression of ovarian somatic cells have been previously attributed to female age. We demonstrate that 407 genes are differentially expressed (FDR&amp;lt;0.05) between normo- and hyporesponder IVF patients after age adjustment. These genes were enriched into pathways of extracellular matrix reorganization (31 genes, FDR&amp;lt;0.005), post-translational protein phosphorylation (16 genes, FDR&amp;lt;0.006) and regulation of insulin-like growth factor transport and uptake by insulin-like growth factor binding proteins (16 genes, FDR&amp;lt;0.019). In addition, the sequencing of &amp;gt; 24 000 individual cells from 3 normoresponder patients revealed the somatic cell types of the preovulatory follicle in high resolution indicating the presence of immune cell populations (N = 4), epithelial cells, theca, cumulus cells and mural granulosa cell sub-populations (N = 7). Bioinformatic cell type deconvolution demonstrated significant differences between proportions of distinct cell sub-populations between normo- and hyporesponder patients and that several differentially expressed genes between the study groups can be attributed to a specific mural granulosa sub-population. Limitations, reasons for caution The number of study participants is small due to the high cost of the study methods. Wider implications of the findings The study demonstrates that hyporesponse to stimulation is associated with age-unrelated disturbances in gene expression of preovulatory somatic cells and that different somatic cell populations may have important underlying functions in the ovarian etiologies of infertility. Trial registration number not applicable

Female Infertility Is Associated with an Altered Expression Profile of Different Members of the Tachykinin Family in Human Granulosa Cells.

Neurokinin B (NKB) and its cognate receptor, NK3R, play a key role in the regulation of reproduction. NKB belongs to the family of tachykinins, which also includes substance P and neurokinin A, both encoded by the by the gene TAC1, and hemokinin-1, encoded by the TAC4 gene. In addition to NK3R, tachykinin effects are mediated by NK1R and NK2R, encoded by the genes TACR1 and TACR2, respectively. The role of these other tachykinins and receptors in the regulation of women infertility is mainly unknown. We have analyzed the expression profile of TAC1, TAC4, TACR1, and TACR2 in mural granulosa and cumulus cells from women presenting different infertility etiologies, including polycystic ovarian syndrome, advanced maternal age, low ovarian response, and endometriosis. We also studied the expression of MME, the gene encoding neprilysin, the most important enzyme involved in tachykinin degradation. Our data show that TAC1, TAC4, TACR1, TACR2, and MME expression is dysregulated in a different manner depending on the etiology of women infertility. The abnormal expression of these tachykinins and their receptors might be involved in the decreased fertility of these patients, offering a new insight regarding the diagnosis and treatment of women infertility.

The granulosa cell response to luteinizing hormone is partly mediated by YAP1-dependent induction of amphiregulin

BackgroundThe LH surge is a pivotal event that triggers multiple key ovarian processes including oocyte maturation, cumulus expansion, follicular wall rupture and luteinization of mural granulosa and theca cells. Recently, LH-dependent activation of the Hippo signaling pathway has been shown to be required for the differentiation of granulosa cells into luteal cells. Still, the precise interactions between Hippo and LH signaling in murine granulosa cells remain to be elucidated.MethodsTo detect the expression of effectors of the Hippo pathway, western blot, immunohistochemical and RT-qPCR analyses were performed on granulosa cells treated with LH in vitro or isolated from immature mice treated with eCG and hCG. Cultured granulosa cells were pretreated with pharmacologic inhibitors to identify the signaling pathways involved in Hippo regulation by LH. To study the roles of Yap1 and Taz in the regulation of the LH signaling cascade, RT-qPCR and microarray analyses were done on granulosa cells from Yap1f/f;Tazf/f mice treated with an adenovirus to drive cre expression. RT-qPCR was performed to evaluate YAP1 binding to the Areg promoter following chromatin immunoprecipitation of granulosa cells collected from mice prior to or 60 min following hCG treatment.ResultsGranulosa cells showed a transient increase in LATS1, YAP1 and TAZ phosphorylation levels in response to the ovulatory signal. This Hippo activation by LH was mediated by protein kinase A. Furthermore, Yap1 and Taz are required for the induction of several LH target genes such as Areg, Pgr and Ptgs2, and for the activation of the ERK1/2 pathway. Consistent with these results, there was a substantial overlap between genes that are upregulated by LH and those that are downregulated following loss of Yap1/Taz, highlighting a major role for Hippo in mediating LH actions in the ovulation process. Finally, we showed that there is a marked recruitment of YAP1 to the Areg promoter of granulosa cells in response to hCG stimulation.ConclusionsOverall, these results indicate that Hippo collaborates with the cAMP/PKA and ERK1/2 pathways to participate in the precise regulation of the LH cascade, and that Areg, as a direct transcriptional target of YAP1, is involved in mediating its actions in the ovary.ASENsG98Xz2ZVCPzJnBcA_Video

Elevated Levels of Follicular Fatty Acids Induce Ovarian Inflammation via ERK1/2 and Inflammasome Activation in PCOS.

Polycystic ovary syndrome (PCOS) is accompanied by chronic inflammation and metabolic disorders. Whether metabolic abnormalities affect inflammation in PCOS or not, the underlying mechanism remains to be clarified. We aimed to investigate changes in fatty acids and their effects on inflammatory response in the follicular niche of PCOS patients. This study recruited 50 PCOS patients and 50 age-matched controls for follicular fluids and ovarian mural granulosa cells collection. The human ovarian granulosa cell line KGN was used for evaluating the effect of oleic acid (OA) stimulation. The levels of follicular fatty acids were measured by liquid chromatography-tandem mass spectrometry. The concentrations of inflammatory cytokines were detected by electrochemiluminescence and enzyme-linked immunosorbent assays. The regulation of inflammation-related genes was confirmed by quantitative polymerase chain reaction and Western blotting after OA stimuli. Three saturated fatty acids and 8 unsaturated fatty acids were significantly elevated in follicular fluids of PCOS patients compared to those in controls. The concentrations of follicular interleukin (IL)-6, IL-8, and mature IL-18 were significantly higher in the PCOS group and were positively correlated with the levels of fatty acids. Moreover, OA stimulation upregulated the transcription levels of IL-6 and IL-8 via extracellularly regulated kinase 1/2 signaling pathways in KGN cells. Furthermore, OA treatment induced reactive oxygen species production and inflammasome activation, which is manifested by enhanced caspase-1 activity and mature IL-18 protein level. Fatty acid metabolism was significantly altered in the follicular niche of PCOS patients. Elevated levels of fatty acids could induce ovarian inflammation both at the transcriptional level and in posttranslational processing.

Ovarian granulosa cells from women with PCOS express low levels of SARS-CoV-2 receptors and co-factors.

PurposeSevere acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection is global pandemic with more than 5 million deaths so far. Female reproductive tract organs express coronavirus-associated receptors and factors (SCARFs), suggesting they may be susceptible to SARS-CoV-2 infection; however, the susceptibility of ovary/follicle/oocyte to the same is still elusive. Co-morbidities like obesity, type-2 diabetes mellitus, cardiovascular disease, etc. increase the risk of SARS-CoV-2 infection. These features are common in women with polycystic ovary syndrome (PCOS), warranting further scope to study SCARFs expression in ovary of these women.Materials and methodsSCARFs expression in ovary and ovarian tissues of women with PCOS and healthy women was explored by analyzing publically available microarray datasets. Transcript expressions of SCARFs were investigated in mural and cumulus granulosa cells (MGCs and CGCs) from control and PCOS women undergoing in vitro fertilization (IVF).ResultsMicroarray data revealed that ovary expresses all genes necessary for SARS-CoV-2 infection. PCOS women mostly showed down-regulated/unchanged levels of SCARFs. MGCs and CGCs from PCOS women showed lower expression of receptors ACE2, BSG and DPP4 and protease CTSB than in controls. MGCs showed lower expression of protease CTSL in PCOS than in controls. Expression of TMPRSS2 was not detected in both cell types.ConclusionHuman ovarian follicle may be susceptible to SARS-CoV-2 infection. Lower expression of SCARFs in PCOS indicates that the risk of SARS-CoV-2 infection to the ovary may be lesser in these women than controls. This knowledge may help in safe practices at IVF settings in the current pandemic.Supplementary InformationThe online version contains supplementary material available at 10.1007/s00404-022-06567-4.

Expression and regulation of estrogen receptor 2 and its coregulators in mouse granulosa cells.

The cooperative effects of estrogen and oocyte-derived paracrine factors (ODPFs) play critical roles in the normal development of ovarian follicles; however, the mechanism underlying this cooperation has not been well studied. The present study aimed to determine whether ODPFs affect estrogen signaling by regulating the expression of estrogen receptor (ESR) and its coregulators in mouse granulosa cells. Some transcripts encoding ESR coregulators were differentially expressed between cumulus and mural granulosa cells (MGCs). The transcript levels of ESR coregulators, including nuclear receptor corepressor 1 and activator 2, in cumulus cells were significantly suppressed by ODPFs; however, they increased when cumulus cell-oocyte complexes were treated with the transforming growth factor beta receptor I inhibitor, SB431542. Moreover, MGCs exhibited significantly higher ESR2 protein and transcript levels than those in cumulus cells. ODPFs promoted Esr2 expression in cumulus cells but had no effect on that in MGCs. Overall, regulation of the expression of ESR2 and its coregulators in cumulus cells by oocytes seems to be one of the mechanisms underlying estrogen-oocyte cooperation in well-developed antral follicles in mice.

Hsa-mir-548 family expression in human reproductive tissues

BackgroundHsa-miR-548ba expressed in ovarian granulosa cells targets PTEN and LIFR, which are essential for ovarian follicle activation and growth. The expression pattern of hsa-miR-548ba correlates with its host gene follicle-stimulating hormone receptor (FSHR), and FSH has a positive influence on hsa-miR-548ba expression. However, hsa-miR-548ba is a member of a large hsa-mir-548 family with potentially overlapping targets. The current study aims to investigate the co-expression of hsa-mir-548 family members in FSHR-positive reproductive tissues and to explore the potential co-regulation of pathways.ResultsFor the above-described analysis, small RNA sequencing data from public data repositories were used. Sequencing results revealed that hsa-miR-548ba was expressed at the highest level in the ovarian granulosa cells and uterine myometrial samples together with another twelve and one hsa-miR-548 family members, respectively. Pathway enrichment analysis of microRNA targets in the ovarian samples revealed the hsa-miR-548ba and hsa-miR-548b-5p co-regulation of RAB geranylgeranylation in mural granulosa cells. Moreover, other hsa-mir-548 family members co-regulate pathways essential for ovarian functions (PIP3 activates AKT signalling and signalling by ERBB4). In addition to hsa-miR-548ba, hsa-miR-548o-3p is expressed in the myometrium, which separately targets the peroxisome proliferator-activated receptor alpha (PPARA) pathway.ConclusionThis study reveals that hsa-mir-548 family members are expressed in variable combinations in the reproductive tract, where they potentially fulfil different regulatory roles. The results provide a reference for further studies of the hsa-mir-548 family role in the reproductive tract.

The Function of Cumulus Cells in Oocyte Growth and Maturation and in Subsequent Ovulation and Fertilization.

Cumulus cells (CCs) originating from undifferentiated granulosa cells (GCs) differentiate in mural granulosa cells (MGCs) and CCs during antrum formation in the follicle by the distribution of location. CCs are supporting cells of the oocyte that protect the oocyte from the microenvironment, which helps oocyte growth and maturation in the follicles. Bi-directional communications between an oocyte and CCs are necessary for the oocyte for the acquisition of maturation and early embryonic developmental competence following fertilization. Follicle-stimulation hormone (FSH) and luteinizing hormone (LH) surges lead to the synthesis of an extracellular matrix in CCs, and CCs undergo expansion to assist meiotic resumption of the oocyte. The function of CCs is involved in the completion of oocyte meiotic maturation and ovulation, fertilization, and subsequent early embryo development. Therefore, understanding the function of CCs during follicular development may be helpful for predicting oocyte quality and subsequent embryonic development competence, as well as pregnancy outcomes in the field of reproductive medicine and assisted reproductive technology (ART) for infertility treatment.

Reproductive senescence impairs the energy metabolism of human luteinized granulosa cells

Research questionFemale age is the single greatest factor influencing reproductive performance and granulosa cells are considered as potential biomarkers of oocyte quality. Is there an age effect on the energy metabolism of human mural granulosa cells? DesignObservational prospective cohort and experimental study including 127 women who had undergone IVF cycles. Women were allocated to two groups: a group of infertile patients aged over 38 years and a control group comprising oocyte donors aged less than 35 years. Individuals with pathologies that could impair fertility were excluded from both groups. Following oocyte retrieval, cumulus and granulosa cells were isolated and their bioenergetic properties (oxidative phosphorylation parameters, rate of aerobic glycolysis and adenine nucleotide concentrations) were analysed and compared. ResultsHuman mural luteinized granulosa and cumulus cells present high rates of aerobic glycolysis that cannot be increased further when mitochondrial ATP synthesis is inhibited. Addition of follicular fluid to the experimental media is necessary to reach the full respiratory capacity of the cells. Granulosa cells from aged women present lower mitochondrial respiration (12.8 ± 1.6 versus 11.2 ± 1.6 pmol O2/min/mg; P = 0.046), although mitochondrial mass is not decreased, and lower aerobic glycolysis, than those from young donors (12.9 ± 1.3 versus 10.9 ± 0.5 mpH/min/mg; P = 0.009). The concurrent decrease in the two energy supply pathways leads to a decrease in the cellular energy charge (0.87 ± 0.01 versus 0.83 ± 0.02; P < 0.001). ConclusionsHuman mural luteinized granulosa cells exhibit a reduction in their energy metabolism as women age that is likely to influence female reproductive potential.

The Differential Metabolomes in Cumulus and Mural Granulosa Cells from Human Preovulatory Follicles

This study evaluated the differences in metabolites between cumulus cells (CCs) and mural granulosa cells (MGCs) from human preovulatory follicles to understand the mechanism of oocyte maturation involving CCs and MGCs. CCs and MGCs were collected from women who were undergoing in vitro fertilization (IVF)/intracytoplasmic sperm injection (ICSI) treatment. The differences in morphology were determined by immunofluorescence. The metabolomics of CCs and MGCs was measured by liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) followed by quantitative polymerase chain reaction (qPCR) and western blot analysis to further confirm the genes and proteins involved in oocyte maturation. CCs and MGCs were cultured for 48 h in vitro, and the medium was collected for detection of hormone levels. There were minor morphological differences between CCs and MGCs. LC-MS/MS analysis showed that there were differences in 101 metabolites between CCs and MGCs: 7 metabolites were upregulated in CCs, and 94 metabolites were upregulated in MGCs. The metabolites related to cholesterol transport and estradiol production were enriched in CCs, while metabolites related to antiapoptosis were enriched in MGCs. The expression of genes and proteins involved in cholesterol transport (ABCA1, LDLR, and SCARB1) and estradiol production (SULT2B1 and CYP19A1) was significantly higher in CCs, and the expression of genes and proteins involved in antiapoptosis (CRLS1, LPCAT3, and PLA2G4A) was significantly higher in MGCs. The level of estrogen in CCs was significantly higher than that in MGCs, while the progesterone level showed no significant differences. There are differences between the metabolomes of CCs and MGCs. These differences may be involved in the regulation of oocyte maturation.

P–178 Mural granulosa cells of the human follicles indirectly show death molecular signals not depending on different ovarian stimulation protocols

Abstract Study question Could the expression of the anti-apoptotic molecules AKT, p-Akt and ERK1/2 in Mural Granulosa Cells (MGC) be considered as marker of oocyte quality? Summary answer MGCs activate cell death pathways in analyzed follicles and it is not influenced by different stimulation protocols and it is not correlated to oocyte competence. What is known already It has been previously demonstrated that apoptosis rate of mural granulosa or cumulus cells (CC) were correlated with follicular oocyte number, age, embryo numbers in IVF/ICSI and also clinical pregnancy. Moreover, our previous data demonstrated that in selected patients, who received recombinant LH associated with recombinant FSH (rFSH), the DNA fragmentation in cumulus cells was significantly lower and pregnancy rate was higher, compared to patients treated with rFSH alone. However, to date little is known about the differences between MGC and CC regarding death/survival pathways and whether the two cell types respond in the same way. Study design, size, duration Molecular study on MGCs to investigate the role of the surviving/apoptotic molecules AKT, p-Akt and ERK1/2 and their relationship with the administration of exogenous r-LH combined with r-FSH administration in ovarian stimulation comparing with r-FSH alone. We analyzed also the oocyte competence, for each follicle, according to the embryo development during in vitro culture and the pregnancy outcome. We included fifty-three normo-responder women undergoing ICSI in two years. Participants/materials, setting, methods Patients were divided into two groups: 1) 34 women were stimulated with r-FSH and used as control group, 2) 19 women were stimulated with r-FSH combined with r-LH. Mural granulosa cells isolated singularly from 255 MII oocytes of the 53 patients were used for the study. The study was conducted in public university laboratory. MGCs obtained from each single follicle were suspended in medium, without serum. For immunoreaction anti-AKT, p-AKT, ERK1/2 antibodies were used. Main results and the role of chance Out of 255 MII oocytes collected, 197 were fertilized and the derived embryos had the following evolution: 117 transferred, 57 vitrified and 23 arrested during in vitro culture. 58 oocytes were not analyzed because of failed fertilization or because of their immature condition (GV or MI). In the MGCs isolated from the follicle of each oocyte generating an embryo, the expression AKT, pAKT and ERK1/2 was analyzed and associated with embryo quality and pregnancy outcomes. Immunoblot analysis on granulose cells showed no statistically significant differences in protein level in MGCs isolated from oocytes that have generated transferred embryos (blastocyst at day 5 or 6) comparing with embryos who arrested during in vitro culture. No differences were found also in MGCs collected from the follicles derived from r-FSH ovarian stimulation compared to r-FSH+r-LH. Moreover, no difference was highlighted even between protein level and pregnancy outcomes. The results seem to demonstrate that the MGCs primarily have an endocrine function and support the growth of the follicle, and finally follow a specific death pathway. This condition is not influenced by different ovarian stimulation protocol, in contrast with CC, and is not correlated to oocyte competence, embryo quality and clinical outcomes. Limitations, reasons for caution Only a limited number of patients have been observed. Wider implications of the findings: Our current and past results suggest that the evaluation of cumulus cells and mural granulosa cells in the same follicle show different expression of molecules involved in the apoptotic pathway and therefore they cannot be used, as molecular markers, in the same way, to assess the competence of oocytes. Trial registration number Not applicable

P–514 RAN-Translation in Fragile X associated Premature Ovarian Insufficiency (FXPOI): FMRpolyG as a predictive tool

Abstract Study question Does repeat-associated non-AUG (RAN) translation lead to accumulation of polyglycine- containing protein (FMRpolyG) in human lymphocytes and mural granulosa cells of FMR1 premutation carriers? Summary answer Lymphocytes and granulosa cells from FMR1 premutation carriers contain intracellular inclusions that stain positive for both FMRpolyG and ubiquitin. What is known already: Fragile-X-associated-Primary-Ovarian-Insufficiency (FXPOI) is characterized by oligo/amenorrhea and hypergonadotropic hypogonadism associated with the expansion of CGG-repeats in the 5’UTR of FMR1, called premutation (PM) (n: 55–200). Approximately 20% of women carrying a FMR1-premutation (PM) allele develop FXPOI. RAN-translation dependent on variable CGG-repeat length is hypothesized to cause FXPOI due to the production of a polyglycine-containing FMR1-protein, FMRpolyG. Recently, FMRpolyG inclusions were found in neuronal brain cells of FXTAS patients and stromal cells of the ovary of an FXPOI patient. Study design, size, duration: Lymphocytes and granulosa cells (GCs) from women with PM (6) and women without PM (10) (controls) were analyzed by immunofluorescence (IF) staining for the presence of inclusions positive for ubiquitin and FMRpolyG. Cell lysis and protein extraction samples were subjected to Fluorescent Western Blot (WB) analysis to detect FMRP and FMRpolyG Participants/materials, setting, methods Human GCs were obtained from follicular fluid after oocyte retrieval and lymphocytes were isolated from peripheral blood using Ficoll-Paque. Cells suspended in PBS were adhered to a glass-coverslip placed at the bottom of the 6-well culture plate, via gravity sedimentation. Adhered cells were fixed, IF staining for FMRpolyG and ubiquitin was performed and analyzed by fluorescence microscopy. Fluorescent WB was used to demonstrate the expression of FMRP, FMRpolyG in extracted protein from lymphocytes and GCs. Main results and the role of chance FMRP was successfully detected by fluorescence WB in both lymphocytes and GCs. FMRP is mainly present in cytoplasm and was expressed in greater amount in GCs than in leukocytes. Moreover, FMRP expression was significantly decreased in GCs from FMR1-PM compared with controls. Lymphocytes from PM-carriers and controls were immunostained for FMRpolyG and ubiquitin. In PM-carriers, FMRpolyG was present as aggregates, whereas in controls only a weak signal without inclusions was detectable. The expression pattern of FMRpolyG in GCs was similar to that in lymphocytes with a significant increase in PM-carriers. There, the FMRpolyG-aggregates additionally demonstrated as ubiquitin-positive inclusions. These may resemble the toxic potential of these protein fractions involved the ovarian damage in developing FXPOI. Limitations, reasons for caution More patients are needed to support the present findings. Further investigation into the possible consequences of these FMRpolyG-positive inclusions in PM-carriers is also advisable. Wider implications of the findings: We found for the first time FMRpolyG-accumulation in lymphocytes and GCs from FMR1-PM-carriers in ubiquitin-positive inclusions. Future experiments evaluating consistency in more patients and elucidating the impact on fertility and prospective value for individual ovarian reserve are therefore in preparation. Trial registration number Not applicable

Dysregulation of anti-Mullerian hormone expression levels in mural granulosa cells of FMR1 premutation carriers

FMR1 premutation (55–200 CGG repeats) results in fragile X-associated primary ovarian insufficiency (FXPOI). We evaluated expression levels of folliculogenesis-related mediators, follicle-stimulating hormone (FSH) receptor and anti-Mullerian hormone (AMH), to gain insights into the mechanisms underlying the reduced ovarian function. Mural granulosa cells (MGCs) were collected from FMR1 premutation carriers and noncarriers undergoing IVF treatments. At baseline, MGCs of carriers demonstrated significantly higher mRNA expression levels of AMH (3.5 ± 2.2, n = 12 and 0.97 ± 0.5, n = 17, respectively; p = 0.0003) and FSH receptor (5.6 ± 2.8 and 2.7 ± 2.8, respectively; p = 0.02) and higher AMH protein expression on immunostaining. Accordingly, FMR1 premutation-transfected COV434 cells exhibited higher AMH protein expression than COV434 cells transfected with 20 CGG repeats. We conclude that FMR1 premutation may lead to dysregulation of AMH expression levels, probably due to a compensatory mechanism. Elucidating the pathophysiology of FXPOI may help in early detection of ovarian dysfunction and tailoring IVF treatments to FMR1 premutation carriers.

The Involvement of Lumican in Human Ovulatory Processes.

Based on a previous global transcriptome sequencing project, we hypothesized that Lumican (LUM) might play a role in ovulatory processes. We sought to determine LUM gene expression under various conditions in human preovulatory follicles. The in vitro expression of LUM mRNA in mural (MGCs) and cumulus (CGCs) granulosa cells was characterized using quantitative real-time polymerase chain reaction (qRT-PCR). Immunohistochemical staining was used to identify human LUM expression in follicles at different developmental stages. Cell signaling studies were performed by treating human MGCs with human chorionic gonadotropin (hCG) and both, different stimulators and inhibitors to determine their effect on LUM expression by using qRT-PCR. Cell confluence studies were carried out to study the correlation between LUM expression and follicle cell proliferation. Follicular MGCs and CGCs of women undergoing in vitro fertilization (IVF) procedures due to endometriosis were analyzed for differences in LUM expression patterns by qRT-PCR. LUM mRNA expression was significantly higher in MGCs as compared to CGCs. In CGCs, LUM mRNA was higher in mature metaphase II (MII) oocytes than in germinal vesicle (GV) and metaphase I (MI) oocytes. LUM expression was significantly upregulated in response to hCG in cultured MGCs. Immunohistochemistry of human ovaries revealed LUM was mostly present in MGCs of large preovulatory and postovulatory follicles and absent from primordial follicles. Using pharmacological activators and inhibitors, we demonstrated that LUM induction by luteinizing hormone (LH)/hCG is carried through the mitogen-activated protein kinase (MEK) pathway. LUM expression was induced in high-density cell cultures in a confluence-dependent manner. MGCs from follicles of subjects with endometriosis exhibited reduced mRNA transcription levels compared to control subjects. Our study confirms that LUM is a newly discovered ovulatory gene. LUM might play an important role during the preovulatory period up until ovulation as well as in endometriosis infertility. A better understanding of LUM's role might provide potential new treatment paradigms for some types of female infertility.