Pro-nerve growth factor in the ovary and human granulosa cells

Abstract

Background: Pro-nerve growth factor must be cleaved to generate mature NGF, which was suggested to be a factor involved in ovarian physiology and pathology. Extracellular proNGF can induce cell death in many tissues. Whether extracellular proNGF exists in the ovary and may play a role in the death of follicular cells or atresia was unknown.

Materials and methods: Immunohistochemistry of human and rhesus monkey ovarian sections was performed. IVF-derived follicular fluid and human granulosa cells were studied by RT-PCR, qPCR, Western blotting, ATP- and caspase-assays.

Results and conclusion: Immunohistochemistry of ovarian sections identified proNGF in granulosa cells and Western blotting of human isolated granulosa cells confirmed the presence of proNGF. Ovarian granulosa cells thus produce proNGF. Recombinant human proNGF even at high concentrations did not affect the levels of ATP or the activity of caspase 3/7, indicating that in granulosa cells proNGF does not induce death. In contrast, mature NGF, which was detected previously in follicular fluid, may be a trophic molecule for granulosa cells with unexpected functions. We found that in contrast to proNGF, NGF increased the levels of the transcription factor early growth response 1 and of the enzyme choline acetyl-transferase. A mechanism for the generation of mature NGF from proNGF in the follicular fluid may be extracellular enzymatic cleavage. The enzyme MMP7 is known to cleave proNGF and was identified in follicular fluid and as a product of granulosa cells. Thus the generation of NGF in the ovarian follicle may depend on MMP7.

Figure 1. The NGF precursor proNGF (A) and mature NGF (B) are present in the cells of the follicular wall in human ovaries

Immunohistochemical staining of sections of human ovaries reveals that proNGF is present in cells of a large antral follicle, namely in GCs and theca cells (TCs). Positive staining reaction is indicated by the brown color. Note that the antiserum is specific for the pro-region of the proNGF molecule and does not recognize mature NGF. Bar: 30 μm. Insert control: Result of an experiment in which the proNGF antiserum was pre-adsorbed.

Figure 2. ProNGF and NGF in human GCs

Left panel: Example of a Western blot: ProNGF is detected in human GCs (cells cultured for 2 and 3 days are shown); middle panel: Control blot, in which pre-adsorbed anti-proNGF antiserum was used; right panel: Example of a Western blot, in which an antiserum to NGF was used. Note that this antiserum, as expected, recognizes both NGF and proNGF and that the GCs shown contain abundant proNGF, while NGF was much less abundant.

Figure 3. Stimulation of GCs with proNGF and NGF has no effects on the viability and the apoptosis of human GCs (A and B) and presence of the receptors for NGF and proNGF in human GCs (C – E)

A: Results of ATP assays with human GCs. The stimulation was performed on day 2-3 for 24 h with different concentrations of proNGF and NGF. Staurosporin (10 μM) was used as positive control. Both recombinant proteins did not significantly affect viability of GCs. Each column represents the relative luminescence of four independent experiments (mean ± SEM) per group. Different letters indicate statistically significant differences between the staurosporine-group and the other groups (p< 0.05). B: Results of caspase assays with human GCs. Stimulation was performed on day 2-3 for 24 h with different concentrations of proNGF and NGF and staurosporine (1 μM) as positive control. Neither proNGF nor NGF promoted apoptotic cell death in the human GCs. Each column represents the relative luminescence of three independent experiments (mean ±SEM). Different letters indicate statistically significant differences between the staurosporine-group and the other groups (p< 0.05). C: Result of RT-PCR experiment detecting the receptors for NGF (TrkA and p75^NTR) and for proNGF (p75^NTR and sortilin) in human GCs on different days of culture; β-actin was used as internal standard. Controls were performed without cDNA. D: Western blot of the proNGF receptors in human GCs on different days of culture; preadsorption with sortilin was used as control and β-actin as internal standard. E: Intracellular localization of sortilin in GCs (day 1). Insert as control, in which non-immune serum was used. Bars: 10 μm

Figure 4. EGR1 and CHAT level increase in human GCs after stimulation with NGF

A: Example of a Western blot with human GCs on day 2 of culture; stimulation of human GCs with NGF (50 ng/ml) for 1 h led to an increase in EGR1 levels whereas proNGF stimulation (50 ng/ml) did not. EGR1 pre-adsorption was used as control and β-actin as internal standard. B: Densitometric analysis of the experiments performed on day 2 of human GC culture. Each column represents the ratio of EGR1 to untreated control (mean ± SEM) of four independent experiments. *, p< 0.05 vs. untreated control. C: Example of a Western blot with human GCs on day 2-3 of culture; stimulation of human GCs with NGF (50 ng/ml) for 24 h led to an increase in CHAT protein levels whereas proNGF stimulation (50 ng/ml) has no significant effect on the cells. β-actin was used as internal standard. D: Densitometric analysis of the experiments performed after 24 h stimulation of human GCs on day 2-3 of culture. Each column represents the ratio of CHAT to untreated control (mean ± SEM) of four independent experiments. ***, p < 0.001 vs. untreated control.

Figure 5. Presence of MMP7 in the follicular fluid, in human and Rhesus monkey ovarian sections and in human GCs

A: Result of a Western blot: MMP7 is present in the FF of 5 IVF-patients. B: Immunohistochemical staining of MMP7 in paraffin sections of human and monkey ovarian sections. Note that MMP7 can be detected in GCs and TCs. Bars: 50 μm C: MMP7 is present in human GCs: mRNA (left panel) and protein (right panel). Controls were performed with RNA instead of input cDNA.