In Vitro Assessment of Iron Effect on Porcine Ovarian Granulosa Cells: Secretory Activity, Markers of Proliferation and Apoptosis

Abstract

It would be desirable to expand the existing general knowledge concerning direct action of metals on the ovary. Nevertheless, the results of testing of iron compound on porcine ovarian cells should be interpreted carefully because iron is an essential element which could also induce changes in cellular processes. The aim of this in vitro study was 1) to examine dose-dependent effects of iron on the secretory activity of porcine ovarian granulosa cells, and 2) to outline the potential intracellular mediators mediating these effects. Specifically, we evaluated the effect of iron sulphate on the release of insulin-like growth factor I (IGF-I) and progesterone, as well as the expression of markers of proliferation (cyclin B1) and apoptosis (caspase-3) in porcine ovarian granulosa cells. Concentrations of IGF-I and progesterone were determined by RIA, cyclin B1 and caspase-3 expression by immunocytochemistry (ICC). Our results show a significantly decreased IGF-I secretion by ovarian granulosa cells after iron sulphate addition at the doses 0.5 and 1.0 mg/ml. The iron sulphate additions at doses 0.17 and 1.0 mg/ml had no effect on progesterone secretion. In contrast, iron sulphate addition at doses 0.17-1.0 mg/ml resulted in stimulation of cyclin B1 and caspase-3 expression. In conclusion, the present results indicate a direct effect of iron on 1) secretion of growth factor IGF-I but not steroid hormone progesterone, 2) expression of markers of proliferation (cyclin B1), or 3) apoptosis (caspase-3) of porcine ovarian granulosa cells. These results support an idea that iron could play a regulatory role in porcine ovarian function: hormone release, proliferation and apoptosis.

Bee pollens originating from different species have unique effects on ovarian cell functions

“…These increases in cell proliferation and death suggest that bee pollen can promote ovarian cell turnover. Similar stimulatory actions on cultured ovarian cell turnover has been previously reported: promotion of both proliferation and apoptosis was observed in known regulators of ovarian functions such as follicle stimulating hormone (Sirotkin et al 2014(Sirotkin et al , 2017, leptin (Sirotkin et al 2012), IGF-I and ghrelin (Sirotkin et al 2014), some siRNAs controlling ovarian cell protein kinases (Sirotkin et al 2010), iron (Kolesarova et al 2011b), and the plant isoflavone daidzein (Sirotkin et al 2017) which is present in bee pollen (Denisow and Denisow-Pietrzyk 2016;Cornara et al 2017;Kieliszek et al 2018). Therefore, daidzein is one component that could be responsible for the effects of bee pollen on the ovary.…”

“…Granulosa cells were isolated from the 2 to 5 mm follicles of ovaries of noncycling pubertal Slovakian white gilts that were approximately 180 d of age and originated from the region of Nitra, Slovakia. The cells were then processed and cultured as previously described (Kolesarova et al 2011a(Kolesarova et al , 2011bSirotkin et al 2012Sirotkin et al , 2017. Briefly, the cells were cultured in sterile DMEM/F12 1:1 medium (BioWhittaker TM , Verviers, Belgium) supplemented with 10% foetal calf serum (BioWhittaker TM ) and 1% antibiotic-antimycotic solution (Sigma, St. Louis, MO, USA).…”

“…After two days of culture, cell viability was assessed using a Trypan blue assay. Afterwards, the cells were prepared for immunocytochemical analysis, and the media were stored for use in a subsequent enzyme-linked immunosorbent assay (ELISA), as described previously (Kolesarova et al 2011a(Kolesarova et al , 2011bSirotkin et al 2012Sirotkin et al , 2017.…”

“…BAX (bcl-2-associated X protein) is known to promote apoptosis, so it is used as a cytoplasmic marker of apoptosis (Peña-Blanco and Garc ıa-S aez 2018). The presence of these cellular markers of proliferation and apoptosis was detected by immunocytochemistry, as previously described (Kolesarova et al 2011a(Kolesarova et al , 2011bSirotkin et al 2012Sirotkin et al , 2017. Specifically, monoclonal primary antibodies against these molecules (Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA; diluted 1:500 in PBS) and secondary swine antibodies against mouse IgG labelled with horseradish peroxidase (Servac, Prague, Czech Republic; diluted 1:1000) were used.…”

The reproductive ecology of iron in women

“…Despite the localized production of transferrin within follicular cells, transferrin concentrations in follicular fluid correlates with serum levels, suggesting that global iron status and iron‐binding capacity contributes to total concentrations more than local transferrin synthesis within the ovary does (Briggs et al, ). Later work that tested the dose‐dependent relationship between iron and ovarian cell function in pig cells suggest that iron is involved in hormone secretion and cell proliferation (Kolesarova et al, ). This suggests that iron status may play a role in egg maturation, and thus impact a woman's overall fecundity (Chavarro et al, ).…”