Various cells are localized to appropriate positions in tissues and induced a unique gene expression and functions. In this study, we show for the first time that age-related changes in ovarian hardness and elasticity are linked to abnormal signal transduction affects in secondary follicles. Immature ovaries had lower hardness and elasticity indices, whereas aged ovaries had lower elasticity and higher hardness indices than mature ovaries. Importantly, an optimal hardness and elasticity is important for normal gene expression profile of secondary follicles. We reproduced the ovarian hardness and elasticity using alginate beads with different concentration and viscosities, for three-dimensional culture of secondary follicles. In the aged-ovarian hardness group, follicular diameter decreased significantly and the expression of inflammation-related markers increased significantly. In the immature-ovarian hardness group, follicular diameter increased significantly and the expression of differentiation and proliferation markers increased significantly. The mechanism transmitted the hardness and elasticity to the change of gene expression was that YAP, transmit the physical conditions to nucleus via actin network, was significantly increased in both condition of immature or aged ovary. The abnormal signaling associated with the age-related changes in ovarian hardness and elasticity may aid in the development of treatments for abnormal follicular development in infertility.

Purpose: We demonstrated that briefly treating ovaries with collagenase before exposure to a hyperosmotic solution preserved secondary follicles. However, preserving antral follicles remained difficult. We investigated whether the absence of lipids in warming (thawing) solution is a limiting factor. Methods: Frozen ovary was thawed in a lipid‐containing solution and investigated for the effect on the structure, function, and viability of granulosa cells, which are important for antral follicle function. Results: Lipids incorporated into the granulosa cell membrane protected the distribution of actin structures lining the cell membrane and maintained the expression of paxillin, actin‐related adapter proteins, and YAP, which induces genes critical for granulosa cell function, to support the survivability of granulosa cells in antral follicles. It also preserved the function of frozen ovaries for 3 months after transplantation into the ovarian bursa of recipient hosts, significantly increasing the total number of viable offspring. Conclusions: Lipids—key components of serum—protected the granulosa cell membrane during warming by preventing the efflux of fatty acids for maintenance of proper localization of the actin cytoskeleton and membrane proteins, supporting normal antral follicle function. The novel method combining collagenase pretreatment with a lipid‐containing warming solution offers a promising approach to maintaining the function of cryopreserved ovaries.

Glomerular podocytes act as a part of filtration barrier in the kidney. The activity of this filter is regulated by ionotropic and metabotropic glutamate receptors. Adjacent podocytes can potentially release glutamate into intercellular space; however, little is known about how podocytes release glutamate. Here, we demonstrated vesicular glutamate transporter 3 (VGLUT3)-dependent glutamate release from podocytes. Immunofluorescence analysis revealed that rat glomerular podocytes and an immortal mouse podocyte cell line (MPCs) express VGLUT1 and VGLUT3. Consistent with this finding, quantitative RT-PCR revealed the expression of VGLUT1 and VGLUT3 mRNA in undifferentiated and differentiated MPCs. In addition, the exocytotic proteins vesicle-associated membrane protein 2, synapsin 1, and synaptophysin 1 were present in punctate patterns, and colocalised with VGLUT3 in MPCs. Interestingly, approximately 30% of VGLUT3 colocalised with VGLUT1. By immunoelectron microscopy, VGLUT3 was often observed around clear vesicle-like structures in differentiated MPCs. Differentiated MPCs released glutamate following depolarization with high potassium levels and after stimulation with the muscarinic agonist pilocarpine. Depletion of VGLUT3 in MPCs by RNA interference reduced depolarization-dependent glutamate release. These results strongly suggest that VGLUT3 is involved in glutamatergic signaling in podocytes and maybe a new drug target for various kidney diseases.

Glomerular podocytes act as a part of the filtration barrier in the kidney. The activity of this filter is regulated by ionotropic and metabotropic glutamate receptors. Adjacent podocytes can potentially release glutamate into the intercellular space; however, little is known about how podocytes release glutamate. Here, we demonstrated vesicular glutamate transporter 3 (VGLUT3)-dependent glutamate release from podocytes. Immunofluorescence analysis revealed that rat glomerular podocytes and an immortal mouse podocyte cell line (MPC) express VGLUT1 and VGLUT3. Consistent with this finding, quantitative RT-PCR revealed the expression of VGLUT1 and VGLUT3 mRNA in undifferentiated and differentiated MPCs. In addition, the exocytotic proteins vesicle-associated membrane protein 2, synapsin 1, and synaptophysin 1 were present in punctate patterns and colocalized with VGLUT3 in MPCs. Interestingly, approximately 30% of VGLUT3 colocalized with VGLUT1. By immunoelectron microscopy, VGLUT3 was often observed around clear vesicle-like structures in differentiated MPCs. Differentiated MPCs released glutamate following depolarization with high potassium levels and after stimulation with the muscarinic agonist pilocarpine. The depletion of VGLUT3 in MPCs by RNA interference reduced depolarization-dependent glutamate release. These results strongly suggest that VGLUT3 is involved in glutamatergic signalling in podocytes and may be a new drug target for various kidney diseases.