KLK3/PSA and cathepsin D activate VEGF-C and VEGF-D.

Vascular endothelial growth factor-C (VEGF-C) acts primarily on endothelial cells, but also on non-vascular targets, for example in the CNS and immune system. Here we describe a novel, unique VEGF-C form in the human reproductive system produced via cleavage by kallikrein-related peptidase 3 (KLK3), aka prostate-specific antigen (PSA). KLK3 activated VEGF-C specifically and efficiently through cleavage at a novel N-terminal site. We detected VEGF-C in seminal plasma, and sperm liquefaction occurred concurrently with VEGF-C activation, which was enhanced by collagen and calcium binding EGF domains 1 (CCBE1). After plasmin and ADAMTS3, KLK3 is the third protease shown to activate VEGF-C. Since differently activated VEGF-Cs are characterized by successively shorter N-terminal helices, we created an even shorter hypothetical form, which showed preferential binding to VEGFR-3. Using mass spectrometric analysis of the isolated VEGF-C-cleaving activity from human saliva, we identified cathepsin D as a protease that can activate VEGF-C as well as VEGF-D.

High-throughput analysis of ovarian granulosa cell transcriptome.

The quality of follicular oocytes depends on interactions with surrounding granulosa cells. Development of molecular techniques and methods enables better understanding of processes underlying mammalian reproduction on cellular level. The success in reproductive biology and medicine in different species depends on reliable assessment of oocyte and embryo viability which presently mainly bases on embryo morphology. Although successful pregnancies have been achieved using this approach, its precision still should be improved and completed with other, more objective, and accurate assessment strategies. Global profiling of gene expression in follicular cumulus cells using microarrays is continuously leading to the establishment of new biomarkers which can be used to select oocytes with highest developmental potential. Even more potential applications and greater precision could be achieved using next generation sequencing (NGS) of granulosa and cumulus cell RNA (RNA-seq). However, due to the high cost, this method is not used as frequently as microarrays at the moment. In any case, high-throughput technologies offer the possibilities and advantages in ovarian somatic cell analysis on scale that has not been noted so far. The aim of this work is to present current directions and examples of global molecular profiling of granulosa cells and underline its impact on reproductive biology and medicine.

Changes in neural network homeostasis trigger neuropsychiatric symptoms.

The mechanisms that regulate the strength of synaptic transmission and intrinsic neuronal excitability are well characterized; however, the mechanisms that promote disease-causing neural network dysfunction are poorly defined. We generated mice with targeted neuron type-specific expression of a gain-of-function variant of the neurotransmitter receptor for glycine (GlyR) that is found in hippocampectomies from patients with temporal lobe epilepsy. In this mouse model, targeted expression of gain-of-function GlyR in terminals of glutamatergic cells or in parvalbumin-positive interneurons persistently altered neural network excitability. The increased network excitability associated with gain-of-function GlyR expression in glutamatergic neurons resulted in recurrent epileptiform discharge, which provoked cognitive dysfunction and memory deficits without affecting bidirectional synaptic plasticity. In contrast, decreased network excitability due to gain-of-function GlyR expression in parvalbumin-positive interneurons resulted in an anxiety phenotype, but did not affect cognitive performance or discriminative associative memory. Our animal model unveils neuron type-specific effects on cognition, formation of discriminative associative memory, and emotional behavior in vivo. Furthermore, our data identify a presynaptic disease-causing molecular mechanism that impairs homeostatic regulation of neural network excitability and triggers neuropsychiatric symptoms.

Regulation of telomerase activity in ovarian granulosa cells.

Follicular development is characterized by intensive proliferation and differentiation of granulosa cells. It was reported that during follicular growth granulosa cells arise from the population of stem cells. One of the main evidences for stem characteristics of the cell is the ability to express telomerase--an enzyme complex responsible for integrity and stability of chromosome ends (telomeres). It was demonstrated that telomerase activity in granulosa cells is linked to their proliferation and differentiation status and is under the control of growth factors and steroid hormones. In this review current knowledge on the existence and regulation of telomerase activity in granulosa cells has been presented.

Telomerase activity in pig granulosa cells proliferating and differentiating in vitro.

The aim of the work was to analyze the telomerase activity (TA) in two different populations of pig granulosa cells (GC) proliferating and differentiating in vitro: (a) in relatively undifferentiated granulosa cells isolated from small (1-2 mm) antral follicles and (b) in functionally advanced, differentiated cells obtained from large (5-7 mm) antral follicles. The proliferative potential in vitro of small follicle granulosa cells (SF-GC) was higher than that of large follicle granulosa cells (LF-GC). EGF stimulated significantly (p<0.01) proliferation in SF-GC as well as LF-GC. FSH did not have a stimulating effect on proliferation in both of the GC populations. Steroidogenesis was induced in both SF- and LF-GC in vitro. Significantly higher (p<0.01) levels of estradiol were measured in LF-GC cultures. In SF-GC, no significantly different effects of EGF and FSH on estradiol production were found. The production of progesterone in vitro was higher in LF-GC than in SF-GC and its production was specifically promoted by FSH in contrast to estradiol the synthesis of which in vitro was less dependent on culture conditions. Using the TRAP assay telomerase activity was detected in freshly isolated and in vitro cultured pig SF- and LF-GC. In EGF, but not FSH stimulated SF-GC, significantly enhanced (p<0.05) TA in comparison with the control was observed at an interval of 24 h of culture. After the 48 h in vitro, levels of TA in both EGF and FSH treated cells were comparable with control. In LF-GC, both EGF and FSH stimulated significantly (p<0.05) TA after the 24h of in vitro culture. At an interval of 48 h, no significant differences in the level of TA were observed between control, EGF and FSH stimulated LF-GC. Comparing the levels of TA in SF- and LF-GC, significantly higher levels of TA were found in control (p<0.05) and EGF (p<0.01) treated SF-GC after 24 h in vitro. On the other hand, absolutely, but not significantly, higher levels of TA were found in LF-GC versus SF-GC in all culture conditions after 48 h in vitro.

Immunohistochemical localization of proliferating cell nuclear antigen (PCNA) in the pig ovary.

The aim of the study was to determine the expression of proliferating cell nuclear antigen protein (PCNA) in the pig ovary. The localization of PCNA was demonstrated in paraffin sections of pig ovarian tissue using primary mouse monoclonal anti-PCNA antibody. In primordial follicles, no remarkable staining for PCNA either in granulosa cells or in the oocytes was observed. In primary to secondary follicles, positive staining in oocytes and in some granulosa cells was detected. The advanced preantral and particularly actively growing small to large antral follicles showed extensive PCNA labeling in the layers of granulosa and theca cells and in the cumulus cells encircling the oocyte. PCNA labeling was expressed in nuclei of oocytes in preantral and small antral follicles. In atretic follicles, the level of PCNA protein expression was dependent on the stage of atresia. Follicles demonstrating advanced atresia showed only limited or no PCNA labeled granulosa and theca cells. The results of the study demonstrate that follicular growth and development in pig ovary may be effectively monitored by determining the granulosa cell expression of PCNA.