Reduced oxygen concentrations regulate the phenotype and function of human granulosa cells in vitro and cause a diminished steroidogenic but increased inflammatory cellular reaction.

Oxygen (O2) concentrations have recently been discussed as important regulators of ovarian cells. Human IVF-derived granulosa cells (human GCs) can be maintained in vitro and are a widely used cellular model for the human ovary. Typically, GCs are cultured at atmospheric O2 levels (approximately around 20%), yet the O2 conditions in vivo, especially in the preovulatory follicle, are estimated to be much lower. Therefore, we comprehensively evaluated the consequences of atmospheric versus hypoxic (1% O2) conditions for 4 days on human GCs. We found lower cellular RNA and protein levels but unchanged cell numbers at 1% O2, indicating reduced transcriptional and/or translational activity. A proteomic analysis showed that 391 proteins were indeed decreased, yet 133 proteins were increased under hypoxic conditions. According to gene ontology (GO) enrichment analysis, pathways associated with metabolic processes, for example amino acid-catabolic-processes, mitochondrial protein biosynthesis, and steroid biosynthesis, were downregulated. Pathways associated with glycolysis, chemical homeostasis, cellular response to hypoxia, and actin filament bundle assembly were upregulated. In accordance with lower CYP11A1 (a cholesterol side-chain cleavage enzyme) levels, progesterone release was decreased. A proteome profiler, as well as IL-6 and IL-8 ELISA assays, revealed that hypoxia led to increased secretion of pro-inflammatory and angiogenic factors. Immunofluorescence studies showed nuclear localization of hypoxia-inducible factor 1α (HIF1α) in human GCs upon acute (2 h) exposure to 1% O2 but not in cells exposed to 1% O2 for 4 days. Hence, the role of HIF1α may be restricted to initiation of the hypoxic response in human GCs. The results provide a detailed picture of hypoxia-induced phenotypic changes in human GCs and reveal that chronically low O2 conditions inhibit the steroidogenic but promote the inflammatory phenotype of these cells.

TRPV2, a novel player in the human ovary and human granulosa cells.

The cation channel 'transient receptor potential vanilloid 2' (TRPV2) is activated by a broad spectrum of stimuli, including mechanical stretch, endogenous and exogenous chemical compounds, hormones, growth factors, reactive oxygen species, and cannabinoids. TRPV2 is known to be involved in inflammatory and immunological processes, which are also of relevance in the ovary. Yet, neither the presence nor possible roles of TRPV2 in the ovary have been investigated. Data mining indicated expression, for example, in granulosa cells (GCs) of the human ovary in situ, which was retained in cultured GCs derived from patients undergoing medical reproductive procedures. We performed immunohistochemistry of human and rhesus monkey ovarian sections and then cellular studies in cultured GCs, employing the preferential TRPV2 agonist cannabidiol (CBD). Immunohistochemistry showed TRPV2 staining in GCs of large antral follicles and corpus luteum but also in theca, endothelial, and stromal cells. TRPV2 transcript and protein levels increased upon administration of hCG or forskolin. Acutely, application of the agonist CBD elicited transient Ca2+ fluxes, which was followed by the production and secretion of several inflammatory factors, especially COX2, IL6, IL8, and PTX3, in a time- and dose-dependent manner. CBD interfered with progesterone synthesis and altered both the proteome and secretome, as revealed by a proteomic study. While studies are somewhat hampered by the lack of highly specific TRPV2 agonist or antagonists, the results pinpoint TRPV2 as a modulator of inflammation with possible roles in human ovarian (patho-)physiology. Finally, as TRPV2 is activated by cannabinoids, their possible ovarian actions should be further evaluated.

RIPK1 and RIPK3 are positive prognosticators for cervical cancer patients and C2 ceramide can inhibit tumor cell proliferation in vitro.

Introduction: The enzymes Receptor-interacting serine/threonine-protein kinase 1 (RIPK1) und 3 (RIPK3) as well as the protein Mixed lineage kinase domain like pseudokinase (pMLKL) play a role in the signaling cascade of necroptosis. This is a form of programmed cell death which is caspase-independent. High-risk human papilloma virus infection can inhibit necroptosis. Thereby, a persistent infection and consequently the development of cervical cancer can be triggered. Aim of this study was the analysis of the expression of RIPK1, RIPK3 and pMLKL in cervical cancer tissue and the evaluation of its prognostic value on overall survival, progression-free survival and additional clinical parameters. Methods: The expression of RIPK1, RIPK3, and pMLKL in cervical cancer tissue microarrays of n = 250 patients was analyzed immunohistochemically. Further, the effect of C2 ceramide on several cervical cancer cell lines (CaSki, HeLa, SiHa) was examined. C2 ceramide is a biologically active short-chain ceramide that induces necroptosis in human luteal granulosa cells. Results: Significantly longer overall survival and progression-free survival rates could be detected in cervical cancer patients expressing nuclear RIPK1 or RIPK3 alone or simultaneously (RIPK1 and RIPK3). Cell viability and proliferation was reduced through C2 ceramide stimulation of cervical cancer cells. Simultaneous stimulation of C2 ceramide and the pan-caspase inhibitor Z-VAD-fmk, or the RIPK1-inhibitor necrostatin-1, partly reversed the negative effect of C2 ceramide on cell viability. This observation could imply that caspase-dependent and -independent forms of cell death, including necroptosis, can occur. AnnexinV-FITC apoptosis staining induced a significant increase in apoptotic cells in CaSki and SiHa cells. The stimulation of CaSki cells with C2 ceramide led to a significant percentual increase in necrotic/intermediate (dying) cells after stimulation with C2 ceramide. In addition, after stimulation with C2 ceramide, CaSki and HeLa cells live cell imaging showed morphological changes which are common for necroptosis. Discussion: In conclusion, RIPK1 and RIPK3 are independent positive predictors for overall survival and progression-free survival in cervical cancer patients. C2 ceramide can reduce cell viability and proliferation in cervical cancer cells by inducing most likely both apoptosis and necroptosis.

Evidence of a role for cAMP in mitochondrial regulation in ovarian granulosa cells.

In the ovary, proliferation and differentiation of granulosa cells (GCs) drive follicular growth. Our immunohistochemical study in a non-human primate, the Rhesus monkey, showed that the mitochondrial activity marker protein cytochrome c oxidase subunit 4 (COX4) increases in GCs in parallel to follicle size, and furthermore, its intracellular localization changes. This suggested that there is mitochondrial biogenesis and trafficking, and implicates the actions of gonadotropins, which regulate follicular growth and ovulation. Human KGN cells, i.e. granulosa tumour cells, were therefore used to study these possibilities. To robustly elevate cAMP, and thereby mimic the actions of gonadotropins, we used forskolin (FSK). FSK increased the cell size and the amount of mitochondrial DNA of KGN cells within 24 h. As revealed by MitoTracker™ experiments and ultrastructural 3D reconstruction, FSK treatment induced the formation of elaborate mitochondrial networks. H89, a protein kinase A (PKA) inhibitor, reduced the network formation. A proteomic analysis indicated that FSK elevated the levels of regulators of the cytoskeleton, among others (data available via ProteomeXchange with identifier PXD032160). The steroidogenic enzyme CYP11A1 (Cytochrome P450 Family 11 Subfamily A Member 1), located in mitochondria, was more than 3-fold increased by FSK, implying that the cAMP/PKA-associated structural changes occur in parallel with the acquisition of steroidogenic competence of mitochondria in KGN cells. In summary, the observations show increases in mitochondria and suggest intracellular trafficking of mitochondria in GCs during follicular growth, and indicate that they may partially be under the control of gonadotropins and cAMP. In line with this, increased cAMP in KGN cells profoundly affected mitochondrial dynamics in a PKA-dependent manner and implicated cytoskeletal changes.

A rapid and robust method for the cryopreservation of human granulosa cells.

Human primary granulosa cells (GCs) derived from women undergoing oocyte retrieval can be cultured and used as a cellular model for the study of human ovarian function. In vitro, they change rapidly, initially resembling cells of the preovulatory follicle and then cells of the corpus luteum. They are derived from individual patients, whose different medical history, lifestyle and age lead to heterogeneity. Thus, cells can rarely be ideally matched for cellular experiments or, if available, only in small quantities. We reasoned that cryopreservation of human GCs may be helpful to improve this situation. Previous studies indicated the feasibility of such an approach, but low survival of human GCs was reported, and effects on human GC functionality were only partially evaluated. We tested a slow freezing protocol (employing FCS and DMSO) for human GCs upon isolation from follicular fluid. We compared cryopreserved and subsequently thawed cells with fresh, non-cryopreserved cells from the same patients. About 80% of human GCs survived freezing/thawing. No differences were found in cell morphology, survival rate in culture, or transcript levels of mitochondrial (COX4, OPA1, TOMM20), steroidogenic (CYP11A1, CYP19A1) or cell-cell contact genes (GJA1) between the two groups in cells cultured for 1-5 days. A proteomic analysis revealed no statistically significant change in the abundance of a total of 5962 proteins. The two groups produced comparable basal levels of progesterone and responded similarly to hCG with elevation of progesterone. Taken together, our results show this to be a rapid and readily available method for the cryopreservation of human GCs. We anticipate that it will allow future large-scale experiments and may thereby improve cellular studies with human ovarian cells.

Exploring the Ion Channel TRPV2 and Testicular Macrophages in Mouse Testis.

The cation channel TRPV2 is known to be expressed by murine macrophages and is crucially involved in their functionality. Macrophages are frequent cells of the mouse testis, an immune-privileged and steroid-producing organ. TRPV2 expression by testicular macrophages and possible changes associated with age or inflammation have not been investigated yet. Therefore, we studied testes of young adult and old wild-type (WT) and AROM+ mice, i.e., transgenic mice overexpressing aromatase. In these animals, inflammatory changes are described in the testis, involving active macrophages, which increase with age. This is associated with impaired spermatogenesis and therefore AROM+ mice are a model for male infertility associated with sterile inflammation. In WT animals, testicular TRPV2 expression was mapped to interstitial CD206+ and peritubular MHC II+ macrophages, with higher levels in CD206+ cells. Expression levels of TRPV2 and most macrophage markers did not increase significantly in old mice, with the exception of CD206. As the number of TRPV2+ testicular macrophages was relatively small, their possible involvement in testicular functions and in aging in WT mice remains to be further studied. In AROM+ testis, TRPV2 was readily detected and levels increased significantly with age, together with macrophage markers and TNF-α. TRPV2 co-localized with F4/80 in macrophages and further studies showed that TRPV2 is mainly expressed by unusual CD206+MHC II+ macrophages, arising in the testis of these animals. Rescue experiments (aromatase inhibitor treatment and crossing with ERαKO mice) restored the testicular phenotype and also abolished the elevated expression of TRPV2, macrophage and inflammation markers. This suggests that TRPV2+ macrophages of the testis are part of an inflammatory cascade initiated by an altered sex hormone balance in AROM+ mice. The changes in testis are distinct from the described alterations in other organs of AROM+, such as prostate and spleen. When we monitored TRPV2 levels in another immune-privileged organ, namely the brain, we found that levels of TRPV2 were not elevated in AROM+ and remained stable during aging. In the adrenal, which similar to the testis produces steroids, we found slight, albeit not significant increases in TRPV2 in both AROM+ and WT mice, which were associated with age. Thus, the changes in the testis are specific for this organ.

Testicular adenosine acts as a pro-inflammatory molecule: role of testicular peritubular cells.

Extracellular ATP has been described to be involved in inflammatory cytokine production by human testicular peritubular cells (HTPCs). The ectonucleotidases ENTPD1 and NT5E degrade ATP and have been reported in rodent testicular peritubular cells. We hypothesized that if a similar situation exists in human testis, ATP metabolites may contribute to cytokine production. Indeed, ENTPD1 and NT5E were found in situ and in vitro in HTPCs. Malachite green assays confirmed enzyme activities in HTPCs. Pharmacological inhibition of ENTPD1 (by POM-1) significantly reduced pro-inflammatory cytokines evoked by ATP treatment, suggesting that metabolites of ATP, including adenosine, are likely involved. We focused on adenosine and detected three of the four known adenosine receptors in HTPCs. One, A2B, was also found in situ in peritubular cells of human testicular sections. The A2B agonist BAY60-6583 significantly elevated levels of IL6 and CXCL8, a result also obtained with adenosine and its analogue NECA. Results of siRNA-mediated A2B down-regulation support a role of this receptor. In mouse peritubular cells, in contrast to HTPCs, all four of the known adenosine receptors were detected; when challenged with adenosine, cytokine expression levels significantly increased. Organotypic short-term testis cultures yielded comparable results and indicate an overall pro-inflammatory action of adenosine in the mouse testis. If transferable to the in vivo situation, our results may implicate that interference with the generation of ATP metabolites or interference with adenosine receptors could reduce inflammatory events in the testis. These novel insights may provide new avenues for treatment of sterile inflammation in male subfertility and infertility.

Filamin A Orchestrates Cytoskeletal Structure, Cell Migration and Stem Cell Characteristics in Human Seminoma TCam-2 Cells.

Filamins are large dimeric F-actin cross-linking proteins, crucial for the mechanosensitive properties of a number of cell types. Due to their interaction with a variety of different proteins, they exert important regulatory functions. However, in the human testis the role of filamins has been insufficiently explored. Immunohistochemical staining of human testis samples identified filamin A (FLNA) in spermatogonia and peritubular myoid cells. Investigation of different testicular tumor samples indicated that seminoma also express FLNA. Moreover, mass spectrometric analyses identified FLNA as one of the most abundant proteins in human seminoma TCam-2 cells. We therefore focused on FLNA in TCam-2 cells, and identified by co-immunoprecipitation LAD1, RUVBL1 and DAZAP1, in addition to several cytoskeletal proteins, as interactors of FLNA. To study the role of FLNA in TCam-2 cells, we generated FLNA-deficient cells using the CRISPR/Cas9 system. Loss of FLNA causes an irregular arrangement of the actin cytoskeleton and mechanical instability, impaired adhesive properties and disturbed migratory behavior. Furthermore, transcriptional activity of typical stem cell factors is increased in the absence of FLNA. In summary, our data suggest that FLNA is crucially involved in balancing stem cell characteristics and invasive properties in human seminoma cells and possibly human testicular germ cells.

A Role for H2O2 and TRPM2 in the Induction of Cell Death: Studies in KGN Cells.

Recent studies showed that KGN cells, derived from a human granulosa cell tumor (GCT), express NADPH oxidase 4 (NOX4), an important source of H2O2. Transient receptor potential melastatin 2 (TRPM2) channel is a Ca2+ permeable cation channel that can be activated by H2O2 and plays an important role in cellular functions. It is also able to promote susceptibility to cell death. We studied expression and functionality of TRPM2 in KGN cells and examined GCT tissue microarrays (TMAs) to explore in vivo relevance. We employed live cell, calcium and mitochondrial imaging, viability assays, fluorescence activated cell sorting (FACS) analysis, Western blotting and immunohistochemistry. We confirmed that KGN cells produce H2O2 and found that they express functional TRPM2. H2O2 increased intracellular Ca2+ levels and N-(p-Amylcinnamoyl)anthranilic acid (ACA), a TRPM2 inhibitor, blocked this action. H2O2 caused mitochondrial fragmentation and apoptotic cell death, which could be attenuated by a scavenger (Trolox). Immunohistochemistry showed parallel expression of NOX4 and TRPM2 in all 73 tumor samples examined. The results suggest that GCTs can be endowed with a system that may convey susceptibility to cell death. If so, induction of oxidative stress may be beneficial in GCT therapy. Our results also imply a therapeutic potential for TRPM2 as a drug target in GCTs.

Human testicular peritubular cells, mast cells and testicular inflammation.

In man, the wall of seminiferous tubules forms a testicular compartment, which contains several layers of smooth muscle-like, "myoid", peritubular cells and extracellular matrix. Its architecture and its cellular composition change in male infertility associated with impaired spermatogenesis. Increased deposits of extracellular matrix, changes in the smooth muscle-like phenotype of peritubular cells and accumulation of immune cells, especially mast cells, are among the striking alterations. Taken together, the changes indicate that inflammatory events take place in particular within this compartment. This short review summarises recent studies, which pinpoint possible mechanisms of the interplay between peritubular cells and mast cells, which may contribute to sterile inflammation and impairments of testicular function. These insights are based mainly on cellular studies, for which we used isolated human testicular peritubular cells (HTPCs), and on the examination of human testicular sections. Recent data on immunological properties of peritubular cells, unexpected roles of the extracellular matrix factor, biglycan, which is secreted by peritubular cells and functions of mast cell products (chymase, tryptase and ATP) are presented. We believe that the results may foster a better understanding of peritubular cells, their roles in the human testis and specifically their involvement in infertility.

Ca2+ Signaling and IL-8 Secretion in Human Testicular Peritubular Cells Involve the Cation Channel TRPV2.

Peritubular cells are part of the wall of seminiferous tubules in the human testis and their contractile abilities are important for sperm transport. In addition, they have immunological roles. A proteomic analysis of isolated human testicular peritubular cells (HTPCs) revealed expression of the transient receptor potential channel subfamily V member 2 (TRPV2). This cation channel is linked to mechano-sensation and to immunological processes and inflammation in other organs. We verified expression of TRPV2 in peritubular cells in human sections by immunohistochemistry. It was also found in other testicular cells, including Sertoli cells and interstitial cells. In cultured HTPCs, application of cannabidiol (CBD), a known TRPV2 agonist, acutely induced a transient increase in intracellular Ca2+ levels. These Ca2+ transients could be blocked both by ruthenium red, an unspecific Ca2+ channel blocker, and tranilast (TRA), an antagonist of TRPV2, and were also abolished when extracellular Ca2+ was removed. Taken together this indicates functional TRPV2 channels in peritubular cells. When applied for 24 to 48 h, CBD induced expression of proinflammatory factors. In particular, mRNA and secreted protein levels of the proinflammatory chemokine interleukin-8 (IL-8/CXCL8) were elevated. Via its known roles as a major mediator of the inflammatory response and as an angiogenic factor, this chemokine may play a role in testicular physiology and pathology.