Changes in the organization of the extracellular matrix in ovarian follicles during the preovulatory phase and atresia. An immunofluorescence study.

The distribution of laminin, type IV collagen and fibronectin was studied by immunofluorescence in rat, pig and cow ovarian follicles. The results obtained in the three species investigated were similar. In all the follicles, laminin and type IV collagen were identically localized in the basal lamina (BL) separating the granulosa and the theca layers. In addition, these two proteins were also distributed in the wall of blood vessels of the thecae and ovarian stroma. The staining showed that the BL of primordial and growing follicles was regular and continuous, but underwent striking modifications during ovulation and atresia. In fact, in preovulatory follicles the BL appeared thinner and discontinuous, whereas it was much thickened and ruptured in atretic follicles. Fibronectin was localized mainly in inner granulosa cells of small and medium-sized growing follicles, and as a broad and irregular layer around the cavity of the degenerated follicles. The results show that each stage of follicular growth and involution is associated with a precise and peculiar pattern of distribution of laminin, type IV collagen and fibronectin. The possibility that these proteins play a role in the local control of ovarian follicular dynamics is advanced

StAR Enhances Transcription of Genes Encoding the Mitochondrial Proteases Involved in Its Own Degradation

Steroidogenic acute regulatory protein (StAR) is essential for steroid hormone synthesis in the adrenal cortex and the gonads. StAR activity facilitates the supply of cholesterol substrate into the inner mitochondrial membranes where conversion of the sterol to a steroid is catalyzed. Mitochondrial import terminates the cholesterol mobilization activity of StAR and leads to mounting accumulation of StAR in the mitochondrial matrix. Our studies suggest that to prevent mitochondrial impairment, StAR proteolysis is executed by at least 2 mitochondrial proteases, ie, the matrix LON protease and the inner membrane complexes of the metalloproteases AFG3L2 and AFG3L2: SPG7/paraplegin. Gonadotropin administration to prepubertal rats stimulated ovarian follicular development associated with increased expression of the mitochondrial protein quality control system. In addition, enrichment of LON and AFG3L2 is evident in StAR-expressing ovarian cells examined by confocal microscopy. Furthermore, reporter studies of the protease promoters examined in the heterologous cell model suggest that StAR expression stimulates up to a 3.5-fold increase in the protease gene transcription. Such effects are StAR-specific, are independent of StAR activity, and failed to occur upon expression of StAR mutants that do not enter the matrix. Taken together, the results of this study suggest the presence of a novel regulatory loop, whereby acute accumulation of an apparent nuisance protein in the matrix provokes a mitochondria to nucleus signaling that, in turn, activates selected transcription of genes encoding the enrichment of mitochondrial proteases relevant for enhanced clearance of StAR

Alginate‐Laminin Hydrogel Supports Long‐Term Neuronal Activity in 3D Human Induced Pluripotent Stem Cell‐Derived Neuronal Networks

Abstract For 3D neural cultures durable hydrogels are required, which persist over a long differentiation period and thus enable the maturation of neuronal networks (NN). Here, 3D models based on human induced pluripotent stem cell‐derived neural progenitor cells that are embedded in hydrogels of either pure alginate or alginate functionalized with the extracellular matrix protein laminin 111 (L111) are established. This study analyzes material characteristics such as porosity, L111 distribution and shear viscosity, cell compatibility of hydrogels by measuring viability and cytotoxicity, and neural function by monitoring cell migration, differentiation as well as NN formation and activity on multielectrode arrays. The addition of L111 increases neural migration and enhances differentiation into neurons and astrocytes as well as synaptogenesis in alginate hydrogels. NN formed in hydrogels are electrically active for up to 206 d and L111‐supplementation further increases electrical activity, network maturation, and synchronicity compared to 2D controls and NN grown in pure alginate hydrogels. L111 addition to alginate gels further accelerates recovery of electrical activity after blockage of sodium channels with tetrodotoxin. In conclusion, NN grown in alginate‐L111 hydrogel blends are promising models for future long‐term applications in disease modeling, drug or chemical evaluation

Transcriptional activation of LON Gene by a new form of mitochondrial stress: A role for the nuclear respiratory factor 2 in StAR overload response (SOR)

High output of steroid hormone synthesis in steroidogenic cells of the adrenal cortex and the gonads requires the expression of the steroidogenic acute regulatory protein (StAR) that facilitates cholesterol mobilization to the mitochondrial inner membrane where the CYP11A1/P450scc enzyme complex converts the sterol to the first steroid. Earlier studies have shown that StAR is active while pausing on the cytosolic face of the outer mitochondrial membrane while subsequent import of the protein into the matrix terminates the cholesterol mobilization activity. Consequently, during repeated activity cycles, high level of post-active StAR accumulates in the mitochondrial matrix. To prevent functional damage due to such protein overload effect, StAR is degraded by a sequence of three to four ATP-dependent proteases of the mitochondria protein quality control system, including LON and the m-AAA membranous proteases AFG3L2 and SPG7/paraplegin. Furthermore, StAR expression in both peni-ovulatory ovarian cells, or under ectopic expression in cell line models, results in up to 3-fold enrichment of the mitochondrial proteases and their transcripts. We named this novel form of mitochondrial stress as StAR overload response (SOR). To better understand the SOR mechanism at the transcriptional level we analyzed first the unexplored properties of the proximal promoter of the LON gene. Our findings suggest that the human nuclear respiratory factor 2 (NRF-2), also known as GA binding protein (GABP), is responsible for 88% of the proximal promoter activity, including the observed increase of transcription in the presence of StAR. Further studies are expected to reveal if common transcriptional determinants coordinate the SOR induced transcription of all the genes encoding the SOR proteases. (C) 2015 Elsevier Ireland Ltd. All rights reserved