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-alpha. 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 ER alpha KO 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

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

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