Expression of AMH and SMAD4 in ovarian tissue and granulosa cells of PCOS rats is higher than that of normal rats
Research has verified that the expression of AMH is frequently elevated in patients with PCOS. Furthermore, AMH can influence the expression of downstream genes SMAD4 utilizing the TGF-β signaling pathway. Our hypothesis posited that the levels of AMH and SMAD4 expression in PCOS rats are elevated compared to those in normal rats. To confirm our hypothesis, we initially employed DHEA-induced modeling. The efficacy of the modeling was determined through the observation of the rat's estrous cycle and examination of tissue sections from the ovaries. Analysis of vaginal smears in rats revealed that the control group had a normal estrous cycle, but the DHEA group experienced a markedly disrupted estrous cycle (Fig. 1A). HE staining of rat ovarian tissues revealed the presence of follicles and corpus luteum in both the blank and oil control groups at all stages. Additionally, the granulosa cells exhibited normal morphology and were orderly distributed. There was a suggestion that there was no discernible distinction between the oil control group and the blank control group, and the influence of oil on the experiment was eliminated. However, in the DHEA group on day 10, follicles and corpus luteum were observed at various stages of development, but there was a decrease in the number of follicles in the developmental stage. On day 15, the DHEA group showed an increase in atretic follicles and cystic dilated follicles, as well as a decrease in corpus luteum. Furthermore, on day 20, the DHEA group exhibited significant cystic dilated follicles with fewer granulosa cell layers and corpus luteum. These findings indicate that the rats with PCOS were successfully modeled by day 20 (Fig. 1B).
AMH and SMAD4 expression was further analyzed in serum, tissues, and cells. On days 15 and 20, the levels of T and AMH in the DHEA group rats were greater compared to the oil control group rats, as observed in the serum. However, on day 10, There was no notable disparity in the amount of T and AMH between both groups. Additionally, the levels of SMAD4 were not substantially different between the two groups at any of the periods examined (Fig. 1C). Immunohistochemical analysis of ovarian tissues indicated that AMH expression was limited to granulosa cells, while SMAD4 expression was observed in both granulosa cells and follicular membrane cells (Fig. 1D). The Western blot technique was employed to assess the protein expression of AMH and SMAD4 in the ovarian tissues of rats from each group on days 10, 15, and 20. On day 10, there was no discernible difference in the levels of AMH protein between the DHEA and control groups. However, the expression increased on days 15 and 20. On the other hand, the expression of SMAD4 protein considerably increased on days 10, 15, and 20 (Fig. 1E). It was proposed that the levels of AMH and SMAD4 expression in the ovarian tissues of PCOS rats were elevated compared to those in normal rats.
To eliminate potential interference, we isolated granulosa cells from both PCOS and control rats for in vitro cultivation, taking into account the expression of SMAD4 in follicular membrane cells. The identification of granulosa cell shape and purity was achieved through the utilization of HE staining and FSHR immunofluorescence. HE staining of rat ovarian granulosa cells revealed intact cells that were large, had clear edges, and were uniform in size. The cells were either polygonal or spindle-shaped. Immunofluorescence staining using a specific antibody for FSHR demonstrated that FSHR was expressed specifically in granulosa cells, with a positive expression rate exceeding 90% (Fig. 1F). The expression of AMH and SMAD4 in the two groups was detected using RT-qPCR and western blot techniques. The results showed that compared to the control group, the granulosa cells in the ovary of PCOS rats had higher expressions of AMH and SMAD4 (Fig. 1G).
To examine the abnormal proliferation and apoptosis of ovarian granulosa cells in PCOS rats, we analyzed the levels of PCNA and BAX proteins in both groups. PCNA is a protein that indicates cell proliferation, whereas BAX is a protein that promotes cell apoptosis. The western blot analysis revealed a reduction in the expression of PCNA protein in the ovarian granulosa cells of PCOS rats, whereas the expression of BAX protein was found to be enhanced (Fig. 1G). The western blot analysis revealed a decrease in the expression of PCNA protein and an increase in BAX protein in the ovarian granulosa cells of PCOS rats (Fig. 1G). The aforementioned investigations validated that the levels of AMH and SMAD4 expression were elevated in PCOS rats compared to normal SD rats. Additionally, the proliferation of ovarian granulosa cells was diminished and apoptosis was augmented in PCOS rats.
A high level of AMH increases the expression of SMAD4, hinders the proliferation of granulosa cells, and stimulates apoptosis.
AMH modulates gene expression in subsequent steps of the TGF-β signaling cascade, with SMAD4 being the sole shared mediator in this pathway. Our theory suggests that in typical physiological circumstances, elevated levels of AMH can increase the expression of SMAD4, suppress the growth of granulocytes, and stimulate apoptosis. The study utilized various concentrations (0, 10, 100, 1000ng/ml) of rAMH to provide treatment to control rat ovarian granulosa cells. The findings indicated that the presence of 100 ng/ml of rAMH led to an elevation in SMAD4 protein expression (Fig. 2A). However, there was no notable disparity in SMAD4 protein expression between the groups treated with 10 ng/ml and 1000 ng/ml of rAMH.
To investigate the impact of varying rAMH dosages on apoptosis and cell growth in more detail, we employed CCK-8 assay to measure cell proliferation and flow cytometry to measure cell apoptosis. The results of the CCK-8 cell value-added assay indicated that the optical density (OD) value of the 10ng/ml rAMH group showed a substantial rise, leading to an increase in cell proliferation. Conversely, the OD values of the 100ng/ml and 1000ng/ml rAMH groups exhibited a considerable drop, resulting in inhibition of cell proliferation. Suppressed cellular growth. The flow apoptosis assay results indicated a notable decrease in the apoptosis rate in the 10ng/ml rAMH group, while an increase was observed in the 100 and 1000ng/ml rAMH groups (Fig. 2B). Furthermore, we identified CyclinA, caspase-3, and BCL-2 proteins, respectively. In comparison to the group treated with 10ng/ml of rAMH, the groups treated with 100ng/ml and 1000ng/ml of rAMH exhibited a decrease in protein expression of CyclinA and BCL-2, and an increase in protein expression of caspase-3 (Fig. 2B). According to the findings from CCK-8 and flow cytometry, it was shown that a low concentration (10ng/ml) of rAMH stimulated cell proliferation and prevented cell apoptosis, whereas large concentrations (100ng/ml, 1000ng/ml) of rAMH hindered cell proliferation and encouraged cell apoptosis. The aforementioned results validated that elevated levels of AMH increased the expression of SMAD4 and suppressed the proliferation of granulosa cells while promoting apoptosis.
AMH plays a role in controlling the proliferation and apoptosis of ovarian granulosa cells through the involvement of SMAD4.
Previous research has verified that PCOS rats had elevated levels of AMH and SMAD4. Furthermore, increased amounts of AMH were found to stimulate the production of SMAD4, impede cell proliferation, and facilitate apoptosis. Thus, we postulated that the excessive production of AMH in PCOS rats has a role in controlling the growth of granulosa cells by increasing the expression of SMAD4. To test the aforementioned concept, we employed siRNA to suppress the expression of SMAD4 in this investigation, to observe any changes in granule cell proliferation and the occurrence of apoptosis.
The results from RT-qPCR and Western blot analyses indicated a significant decrease in SMAD4 expression in granule cells following siRNA transfection, demonstrating the successful accomplishment of transfection. The expression of PCNA and BAX proteins in the cells of the knockdown group was compared to that of the negative control group. The Western blot analysis indicated that the cells exhibited an upregulation of PCNA protein expression and a downregulation of BAX protein expression following the suppression of SMAD4 (Fig. 3). The current work definitively established that AMH may play a role in controlling the proliferation of granulosa cells and programmed cell death through SMAD4 in the rat model of PCOS induced by DHEA.